RESUMO
Here, we present a protocol for lentiviral delivery of CRISPR-Cas9 to human induced pluripotent stem cell (iPSC)-derived macrophages using co-incubation with VPX virus-like particles (VPX-VLPs). We describe steps for producing polybrene and puromycin kill curves, VPX viral production, and VPX-VLP titration by western blotting. We then detail procedures for iPSC macrophage precursor lentiviral transduction and lentiviral CRISPR-Cas9-based knockout in iPSC-derived macrophages. This protocol uses efficient genome-editing techniques to explore macrophage involvement in immune response, chronic inflammation, neurodegenerative disease, and cancer progression. For complete details on the use and execution of this protocol, please refer to Navarro-Guerrero et al.1.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doenças Neurodegenerativas , Humanos , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , MacrófagosRESUMO
Identification of the genes and processes mediating genetic association signals for complex diseases represents a major challenge. As many of the genetic signals for type 2 diabetes (T2D) exert their effects through pancreatic islet-cell dysfunction, we performed a genome-wide pooled CRISPR loss-of-function screen in a human pancreatic beta cell line. We assessed the regulation of insulin content as a disease-relevant readout of beta cell function and identified 580 genes influencing this phenotype. Integration with genetic and genomic data provided experimental support for 20 candidate T2D effector transcripts including the autophagy receptor CALCOCO2. Loss of CALCOCO2 was associated with distorted mitochondria, less proinsulin-containing immature granules and accumulation of autophagosomes upon inhibition of late-stage autophagy. Carriers of T2D-associated variants at the CALCOCO2 locus further displayed altered insulin secretion. Our study highlights how cellular screens can augment existing multi-omic efforts to support mechanistic understanding and provide evidence for causal effects at genome-wide association studies loci.
Assuntos
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Humanos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Estudo de Associação Genômica Ampla , Insulina/genética , Células Secretoras de Insulina/metabolismoRESUMO
The tumor suppressor p53 is the most frequently mutated protein in human cancer. The majority of these mutations are missense mutations in the DNA binding domain of p53. Restoring p53 tumor suppressor function could have a major impact on the therapy for a wide range of cancers. Here we report a virtual screening approach that identified several small molecules with p53 reactivation activities. The UCI-LC0023 compound series was studied in detail and was shown to bind p53, induce a conformational change in mutant p53, restore the ability of p53 hotspot mutants to associate with chromatin, reestablish sequence-specific DNA binding of a p53 mutant in a reconstituted in vitro system, induce p53-dependent transcription programs, and prevent progression of tumors carrying mutant p53, but not p53null or p53WT alleles. Our study demonstrates feasibility of a computation-guided approach to identify small molecule corrector drugs for p53 hotspot mutations.
Assuntos
Neoplasias , Proteína Supressora de Tumor p53 , Linhagem Celular Tumoral , Cromatina , DNA , Humanos , Mutação , Neoplasias/tratamento farmacológico , Domínios Proteicos , Proteína Supressora de Tumor p53/metabolismoRESUMO
The bromodomain-containing proteins BRD9 and BRD7 are part of the human SWI/SNF chromatin-remodeling complexes BAF and PBAF. To date, no selective inhibitor for BRD7/9 has been reported despite its potential value as a biological tool or as a lead for future therapeutics. The quinolone-fused lactam LP99 is now reported as the first potent and selective inhibitor of the BRD7 and BRD9 bromodomains. Development of LP99 from a fragment hit was expedited through balancing structure-based inhibitor design and biophysical characterization against tractable chemical synthesis: Complexity-building nitro-Mannich/lactamization cascade processes allowed for early structure-activity relationship studies whereas an enantioselective organocatalytic nitro-Mannich reaction enabled the synthesis of the lead scaffold in enantioenriched form and on scale. This epigenetic probe was shown to inhibit the association of BRD7 and BRD9 to acetylated histones inâ vitro and in cells. Moreover, LP99 was used to demonstrate that BRD7/9 plays a role in regulating pro-inflammatory cytokine secretion.
Assuntos
Proteínas Cromossômicas não Histona/antagonistas & inibidores , Descoberta de Drogas , Lactamas/química , Lactamas/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Humanos , Modelos Moleculares , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
Next-generation sequencing (NGS) has revolutionized genetics and enabled the accurate identification of many genetic variants across many genomes. However, detection of biologically important low-frequency variants within genetically heterogeneous populations remains challenging, because they are difficult to distinguish from intrinsic NGS sequencing error rates. Approaches to overcome these limitations are essential to detect rare mutations in large cohorts, virus or microbial populations, mitochondria heteroplasmy, and other heterogeneous mixtures such as tumors. Modifications in library preparation can overcome some of these limitations, but are experimentally challenging and restricted to skilled biologists. This paper describes a novel quality filtering and base pruning pipeline, called Complex Heterogeneous Overlapped Paired-End Reads (CHOPER), designed to detect sequence variants in a complex population with high sequence similarity derived from All-Codon-Scanning (ACS) mutagenesis. A novel fast alignment algorithm, designed for the specified application, has O(n) time complexity. CHOPER was applied to a p53 cancer mutant reactivation study derived from ACS mutagenesis. Relative to error filtering based on Phred quality scores, CHOPER improved accuracy by about 13% while discarding only half as many bases. These results are a step toward extending the power of NGS to the analysis of genetically heterogeneous populations.
Assuntos
Algoritmos , Análise Mutacional de DNA , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Mutagênese , Estatística como Assunto/métodos , Códon/genética , Biblioteca Gênica , Humanos , Neoplasias/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
The bromodomain-containing proteins BRD9 and BRD7 are part of the human SWI/SNF chromatin-remodeling complexes BAF and PBAF. To date, no selective inhibitor for BRD7/9 has been reported despite its potential value as a biological tool or as a lead for future therapeutics. The quinolone-fused lactam LP99 is now reported as the first potent and selective inhibitor of the BRD7 and BRD9 bromodomains. Development of LP99 from a fragment hit was expedited through balancing structure-based inhibitor design and biophysical characterization against tractable chemical synthesis: Complexity-building nitro-Mannich/lactamization cascade processes allowed for early structure-activity relationship studies whereas an enantioselective organocatalytic nitro-Mannich reaction enabled the synthesis of the lead scaffold in enantioenriched form and on scale. This epigenetic probe was shown to inhibit the association of BRD7 and BRD9 to acetylated histones inâ vitro and in cells. Moreover, LP99 was used to demonstrate that BRD7/9 plays a role in regulating pro-inflammatory cytokine secretion.
RESUMO
BACKGROUND: Asthma guidelines recommend prescription of inhaled corticosteroids at a reduced dosage in children compared to older patients in order to minimize the systemic exposure and risk of unwanted side effects. In children, pressurized metered dose inhalers (pMDI) are recommended in combination with a valved holding chamber (VHC) to overcome the problem of coordinating inhalation with actuation. However, the influence of age and body size on the systemic exposure of drugs to be administered via a pMDI with VHC is still not fully elucidated. Therefore, we aimed to compare the systemic exposure to the active ingredients of a fixed combination of beclometasone-dipropionate/formoterol-fumarate administered via pMDI with VHC in children, adolescents and adults. METHODS: The pharmacokinetics of formoterol and beclometasone-17-monopropionate (active metabolite of beclometasone-dipropionate) was evaluated over 8 h from three studies, each performed in a different age and body size group. Children (7-11 years, n = 20), adolescents (12-17 years, n = 29) and adults (≥18 years, n = 24) received a single dose of beclometasone/formoterol (children: 200 µg/24 µg, adolescents and adults: 400 µg/24 µg) via pMDI with AeroChamber Plus™. RESULTS: The systemic exposure in children in comparison to adolescents was equivalent for formoterol while it was halved for beclometasone-17-monopropionate in accordance with the halved dose of beclometasone administered in children (90% CIs within 0.8-1.25 for formoterol and 0.4-0.625 for beclometasone-17-monopropionate). The systemic exposure to beclometasone-17-monopropionate and formoterol was equivalent between adolescents and adults. CONCLUSIONS: The systemic exposure to the active ingredients of a fixed dose combination of beclometasone/formoterol administered via pMDI with AeroChamber Plus™ correlates with the nominal dose independently of patient age and body size. Thus, dose reduction in relation to age when using a pMDI with VHC may be unnecessary for reducing the systemic exposure in children.
Assuntos
Antiasmáticos/farmacocinética , Asma/tratamento farmacológico , Beclometasona/farmacocinética , Etanolaminas/farmacocinética , Administração por Inalação , Adolescente , Adulto , Fatores Etários , Idoso , Antiasmáticos/administração & dosagem , Beclometasona/administração & dosagem , Criança , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Etanolaminas/administração & dosagem , Feminino , Fumarato de Formoterol , Humanos , Masculino , Inaladores Dosimetrados , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: According to international treatment guidelines, inhaled rapid-acting ß2 agonists should be used for the control of symptoms in patients with asthma. We compared the efficacy and safety of an extrafine combination inhaler containing a corticosteroid (beclometasone) plus a rapid-onset, long-acting ß2 agonist (formoterol) with a short-acting ß2 agonist (salbutamol) as reliever strategies in patients taking beclometasone-formoterol combination as maintenance treatment. METHODS: In a double-blind trial undertaken in 183 centres in 14 European countries over 48 weeks, patients (aged ≥18 years) with asthma that was not fully controlled, with a forced expiratory volume in 1 s (FEV1) of at least 60% predicted, had a 2-week run in. During this period, patients were treated with a combination of beclometasone 100 µg and formoterol 6 µg per one inhalation twice daily plus salbutamol 100 µg as required delivered by use of a pressurised metered-dose inhaler. They were then randomly assigned in a 1:1 ratio with a computer-generated randomisation list to receive beclometasone 100 µg plus formoterol 6 µg or salbutamol 100 µg as reliever in addition to maintenance with beclometasone 100 µg plus formoterol 6 µg twice daily. Primary outcome was the time to first severe exacerbation (admission to hospital or visit to emergency department, or use of systemic steroids for ≥3 consecutive days). Secondary outcomes were number of severe exacerbations (events per 100 patients per year), time to and number of mild exacerbations, additional exacerbation variables, lung function, symptom scores, and asthma control. Analysis was by intention to treat. The study is registered with ClinicalTrials.gov, number NCT00861926. FINDINGS: 1714 patients were randomly assigned to the as-needed beclometasone-formoterol (n=857) and as-needed salbutamol groups (n=857), and 1701 were analysed (852 and 849, respectively). 326 severe exacerbations were reported by 251 patients during the study, and 99 versus 152 patients had at least one exacerbation during the 48 weeks, respectively. Compared with beclometasone-formoterol plus salbutamol as needed, beclometasone-formoterol for both maintenance and reliever treatment significantly increased the time to first exacerbation (209 days vs 134 days) by 75 days, with a 36% reduction in risk (hazard ratio 0·64 [95% CI 0·49 to 0·82]; p=0·0005), and the estimated probability was 12% and 18%, respectively (p=0·0003). The number of days with mild asthma exacerbations was also lower with as-needed beclometasone-formoterol than with as-needed salbutamol (56·04 days per patient per year vs 65·11 days per patient per year; 0·86 [0·76 to 0·98]; p=0·021). From the run-in period to week 48, both treatments improved symptoms (mean change -1·59 [-1·94 to -1·25] in the as-needed beclometasone-formoterol group vs -1·44 [-1·78 to -1·10] in the as-needed salbutamol group, difference -0·15 [-0·60 to 0·30]; p=0·507), percentage of asthma control days (9·5% [7·3 to 11·8] vs 10·9% [8·7 to 13·1], respectively, -1·4 [-4·3 to 1·6]; p=0·359), use of reliever (-0·29 [-0·38 to -0·20] vs -0·27 [-0·36 to -0·19], respectively, -0·02 [-0·13 to 0·10]; p=0·794), and lung function (FEV1, 0·090 [0·060 to 0·120] vs 0·090 [0·060-0·120], respectively, 0·001 [-0·040 to 0·040]; p=0·969), and were well tolerated (patients with serious adverse events, 32 [4%] and 41 [5%], respectively). INTERPRETATION: Our results lend support to the use of the combination of a single inhaled corticosteroid plus a rapid-onset, long-acting ß2 agonist for maintenance and relief in patients with moderate to severe asthma and provide encouraging data for the formulation of beclometasone-formoterol for this use. FUNDING: Chiesi Farmaceutici.
Assuntos
Antiasmáticos/uso terapêutico , Asma/tratamento farmacológico , Beclometasona/uso terapêutico , Etanolaminas/uso terapêutico , Administração por Inalação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Albuterol/administração & dosagem , Albuterol/uso terapêutico , Antiasmáticos/administração & dosagem , Beclometasona/administração & dosagem , Método Duplo-Cego , Combinação de Medicamentos , Etanolaminas/administração & dosagem , Feminino , Volume Expiratório Forçado , Fumarato de Formoterol , Humanos , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento , Adulto JovemRESUMO
The tumour suppressor p53 is the most frequently mutated gene in human cancer. Reactivation of mutant p53 by small molecules is an exciting potential cancer therapy. Although several compounds restore wild-type function to mutant p53, their binding sites and mechanisms of action are elusive. Here computational methods identify a transiently open binding pocket between loop L1 and sheet S3 of the p53 core domain. Mutation of residue Cys124, located at the centre of the pocket, abolishes p53 reactivation of mutant R175H by PRIMA-1, a known reactivation compound. Ensemble-based virtual screening against this newly revealed pocket selects stictic acid as a potential p53 reactivation compound. In human osteosarcoma cells, stictic acid exhibits dose-dependent reactivation of p21 expression for mutant R175H more strongly than does PRIMA-1. These results indicate the L1/S3 pocket as a target for pharmaceutical reactivation of p53 mutants.
Assuntos
Biologia Computacional/métodos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Compostos Aza/farmacologia , Sítios de Ligação , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Cisteína/genética , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Simulação de Dinâmica Molecular , Oxepinas/química , Oxepinas/farmacologia , Estabilidade Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Reprodutibilidade dos Testes , Relação Estrutura-Atividade , Transcrição Gênica/efeitos dos fármacos , Proteína Supressora de Tumor p53/genéticaRESUMO
BACKGROUND: Short-acting beta2-agonists like salbutamol and terbutaline are used as rescue medications for acute bronchoconstriction and relief of symptoms due to their rapid onset of action. The aim of this study was to assess whether inhaled beclomethasone dipropionate (BDP)/formoterol fumarate (FF) combination in extrafine formulation is non-inferior to salbutamol in the speed of reverting methacholine-induced bronchoconstriction and symptoms. METHODS: Fifty-six asthmatic patients were examined in a multicentre, randomised, double blind, double dummy, active treatment and placebo controlled three period cross-over study. On three different days, a single dose of BDP/FF 100/6 µg in pressurised metered-dose inhaler (pMDI) extrafine formulation or salbutamol 200 µg pMDI or placebo was inhaled after FEV(1) had dropped by 30-45% with methacholine challenge. RESULTS: The median time to recovery of FEV(1) to 85% of baseline was similar for BDP/FF and salbutamol (3.66 and 2.15 min, respectively), but significantly longer for placebo (21.1 min). The planned analysis on adjusted mean time to recovery showed that the difference from methacholine-induced bronchoconstriction between BDP/FF and salbutamol was 3.82 min (95% confidence interval: -0.85 to 8.5), therefore greater than 3 min supposed in the study design. The difference between BDP/FF and salbutamol was not clinically significant. The two active treatments were also comparable in terms of the relief of symptoms (as assessed by the Borg dyspnoea scale). CONCLUSIONS: BDP/FF combination has a fast onset of action, similar to that of salbutamol, and may represent a good alternative as rescue medication in asthmatic patients.
Assuntos
Albuterol/uso terapêutico , Asma/tratamento farmacológico , Beclometasona/administração & dosagem , Espasmo Brônquico/tratamento farmacológico , Etanolaminas/administração & dosagem , Cloreto de Metacolina/farmacologia , Adulto , Asma/fisiopatologia , Beclometasona/efeitos adversos , Espasmo Brônquico/induzido quimicamente , Espasmo Brônquico/fisiopatologia , Estudos Cross-Over , Método Duplo-Cego , Quimioterapia Combinada , Etanolaminas/efeitos adversos , Feminino , Volume Expiratório Forçado/efeitos dos fármacos , Fumarato de Formoterol , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
The tumor suppressor protein p53 can lose its function upon single-point missense mutations in the core DNA-binding domain ("cancer mutants"). Activity can be restored by second-site suppressor mutations ("rescue mutants"). This paper relates the functional activity of p53 cancer and rescue mutants to their overall molecular dynamics (MD), without focusing on local structural details. A novel global measure of protein flexibility for the p53 core DNA-binding domain, the number of clusters at a certain RMSD cutoff, was computed by clustering over 0.7 µs of explicitly solvated all-atom MD simulations. For wild-type p53 and a sample of p53 cancer or rescue mutants, the number of clusters was a good predictor of in vivo p53 functional activity in cell-based assays. This number-of-clusters (NOC) metric was strongly correlated (r(2)â=â0.77) with reported values of experimentally measured ΔΔG protein thermodynamic stability. Interpreting the number of clusters as a measure of protein flexibility: (i) p53 cancer mutants were more flexible than wild-type protein, (ii) second-site rescue mutations decreased the flexibility of cancer mutants, and (iii) negative controls of non-rescue second-site mutants did not. This new method reflects the overall stability of the p53 core domain and can discriminate which second-site mutations restore activity to p53 cancer mutants.
Assuntos
Simulação de Dinâmica Molecular , Mutação , Neoplasias/genética , Proteína Supressora de Tumor p53/genética , Humanos , Modelos Moleculares , Ligação Proteica/genética , Conformação ProteicaRESUMO
BACKGROUND: Pathogen recognition by dendritic cells (DC) is crucial for the initiation of both innate and adaptive immune responses. Activation of Toll-like Receptors (TLRs) by microbial molecular patterns leads to the maturation of DC, which present the antigen and activate T cells in secondary lymphoid tissues. Cytokine production by DC is critical for shaping the adaptive immune response by regulating T helper cell differentiation. It was previously shown by our group that Src kinases play a key role in cytokines production during TLR4 activation in human DC. PRINCIPAL FINDINGS: In this work we investigated the role of Src kinases during different TLRs triggering in human monocyte-derived DC (MoDC). We found that Src family kinases are important for a balanced production of inflammatory cytokines by human MoDC upon stimulation of TLR3 and 8 with their respective agonists. Disruption of this equilibrium through pharmacological inhibition of Src kinases alters the DC maturation pattern. In particular, while expression of IL-12 and other inflammatory cytokines depend on Src kinases, the induction of IL-23 and co-stimulatory molecules do not. Accordingly, DC treated with Src inhibitors are not compromised in their ability to induce CD4 T cell proliferation and to promote the Th17 subset survival but are less efficient in inducing Th1 differentiation. CONCLUSIONS: We suggest that the pharmacological modulation of DC maturation has the potential to shape the quality of the adaptive immune response and could be exploited for the treatment of inflammation-related diseases.
Assuntos
Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Interleucina-12/biossíntese , Interleucina-23/biossíntese , Receptores Toll-Like/agonistas , Quinases da Família src/metabolismo , Western Blotting , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Citometria de Fluxo , Humanos , Imidazóis/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Pirimidinas/farmacologia , Receptor 3 Toll-Like/agonistas , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/metabolismo , Receptor 8 Toll-Like/agonistas , Receptor 8 Toll-Like/metabolismo , Receptores Toll-Like/metabolismo , Quinases da Família src/antagonistas & inibidoresRESUMO
In vitro scanning mutagenesis strategies are valuable tools to identify critical residues in proteins and to generate proteins with modified properties. We describe the fast and simple All-Codon Scanning (ACS) strategy that creates a defined gene library wherein each individual codon within a specific target region is changed into all possible codons with only a single codon change per mutagenesis product. ACS is based on a multiplexed overlapping mutagenesis primer design that saturates only the targeted gene region with single codon changes. We have used ACS to produce single amino-acid changes in small and large regions of the human tumor suppressor protein p53 to identify single amino-acid substitutions that can restore activity to inactive p53 found in human cancers. Single-tube reactions were used to saturate defined 30-nt regions with all possible codon changes. The same technique was used in 20 parallel reactions to scan the 600-bp fragment encoding the entire p53 core domain. Identification of several novel p53 cancer rescue mutations demonstrated the utility of the ACS approach. ACS is a fast, simple and versatile method, which is useful for protein structure-function analyses and protein design or evolution problems.
Assuntos
Substituição de Aminoácidos , Códon , Genes Neoplásicos , Genes p53 , Sequência de Bases , Linhagem Celular , Biblioteca Gênica , Humanos , Dados de Sequência Molecular , Mutação , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND: The monoclonal antibody trastuzumab has survival benefit when given with chemotherapy to patients with early, operable, and metastatic breast cancer that has HER2 (also known as ERBB2) overexpression or amplification. We aimed to assess event-free survival in patients with HER2-positive locally advanced or inflammatory breast cancer receiving neoadjuvant chemotherapy with or without 1 year of trastuzumab. METHODS: We compared 1 year of treatment with trastuzumab (given as neoadjuvant and adjuvant treatment; n=117) with no trastuzumab (118), in women with HER2-positive locally advanced or inflammatory breast cancer treated with a neoadjuvant chemotherapy regimen consisting of doxorubicin, paclitaxel, cyclophosphamide, methotrexate, and fluorouracil. Randomisation was done with a computer program and minimisation technique, taking account of geographical area, disease stage, and hormone receptor status. Investigators were informed of treatment allocation. A parallel cohort of 99 patients with HER2-negative disease was included and treated with the same chemotherapy regimen. Primary endpoint was event-free survival. Analysis was by intention to treat. This study is registered, number ISRCTN86043495. FINDINGS: Trastuzumab significantly improved event-free survival in patients with HER2-positive breast cancer (3-year event-free survival, 71% [95% CI 61-78; n=36 events] with trastuzumab, vs 56% [46-65; n=51 events] without; hazard ratio 0.59 [95% CI 0.38-0.90]; p=0.013). Trastuzumab was well tolerated and, despite concurrent administration with doxorubicin, only two patients (2%) developed symptomatic cardiac failure. Both responded to cardiac drugs. INTERPRETATION: The addition of neoadjuvant and adjuvant trastuzumab to neoadjuvant chemotherapy should be considered for women with HER2-positive locally advanced or inflammatory breast cancer to improve event-free survival, survival, and clinical and pathological tumour responses. FUNDING: F Hoffmann-La Roche.
Assuntos
Adenocarcinoma/tratamento farmacológico , Anticorpos Monoclonais/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Receptor ErbB-2/efeitos dos fármacos , Adenocarcinoma/patologia , Adulto , Anticorpos Monoclonais Humanizados , Neoplasias da Mama/patologia , Quimioterapia Adjuvante , Terapia Combinada , Ciclofosfamida/administração & dosagem , Intervalo Livre de Doença , Doxorrubicina/administração & dosagem , Feminino , Fluoruracila/administração & dosagem , Seguimentos , Genes erbB-2/genética , Humanos , Infusões Intravenosas , Metotrexato/administração & dosagem , Pessoa de Meia-Idade , Terapia Neoadjuvante , Paclitaxel/administração & dosagem , Modelos de Riscos Proporcionais , Estudos Prospectivos , Fatores de Tempo , TrastuzumabRESUMO
Many protein engineering problems involve finding mutations that produce proteins with a particular function. Computational active learning is an attractive approach to discover desired biological activities. Traditional active learning techniques have been optimized to iteratively improve classifier accuracy, not to quickly discover biologically significant results. We report here a novel active learning technique, Most Informative Positive (MIP), which is tailored to biological problems because it seeks novel and informative positive results. MIP active learning differs from traditional active learning methods in two ways: (1) it preferentially seeks Positive (functionally active) examples; and (2) it may be effectively extended to select gene regions suitable for high throughput combinatorial mutagenesis. We applied MIP to discover mutations in the tumor suppressor protein p53 that reactivate mutated p53 found in human cancers. This is an important biomedical goal because p53 mutants have been implicated in half of all human cancers, and restoring active p53 in tumors leads to tumor regression. MIP found Positive (cancer rescue) p53 mutants in silico using 33% fewer experiments than traditional non-MIP active learning, with only a minor decrease in classifier accuracy. Applying MIP to in vivo experimentation yielded immediate Positive results. Ten different p53 mutations found in human cancers were paired in silico with all possible single amino acid rescue mutations, from which MIP was used to select a Positive Region predicted to be enriched for p53 cancer rescue mutants. In vivo assays showed that the predicted Positive Region: (1) had significantly more (p<0.01) new strong cancer rescue mutants than control regions (Negative, and non-MIP active learning); (2) had slightly more new strong cancer rescue mutants than an Expert region selected for purely biological considerations; and (3) rescued for the first time the previously unrescuable p53 cancer mutant P152L.
Assuntos
Biologia Computacional/métodos , Análise Mutacional de DNA/métodos , Proteína Supressora de Tumor p53/genética , Algoritmos , Inteligência Artificial , Simulação por Computador , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Modelos Genéticos , Modelos Moleculares , Mutação , Engenharia de Proteínas , Reprodutibilidade dos Testes , Proteína Supressora de Tumor p53/metabolismo , Leveduras/genética , Leveduras/metabolismoRESUMO
It was shown previously that bacterial polysaccharides (PS), which naturally contain both positive and negative charges, are able to activate T cells and APCs. However, the vast majority of bacterial PS are anionic and do not have these properties. In this study, we show that chemical introduction of positive charges into naturally anionic bacterial PS confers to the resulting zwitterionic PS (ZPS) the ability to activate pure human monocytes, monocyte-derived dendritic cells, and mouse bone marrow-derived dendritic cells, as do natural bacterial ZPS. Cells are induced to up-regulate MHC class II and costimulatory molecules and to produce cytokines. In mixed monocyte-T cell cocultures, ZPS induce MHC II-dependent T cell proliferation and up-regulation of activation markers. These stimulatory qualities of ZPS disappear when the positive charge is chemically removed from the molecules and thus the zwitterionic motif is destroyed. The ability of natural and chemically derived ZPS to activate APCs can be blocked by anti-TLR2 mAbs, and TLR2 transfectants show reporter gene transcription upon incubation with ZPS. In conclusion, the generation of a zwitterionic motif in bacterial PS confers the ability to activate both APCs and T cells. This finding has important implications for the design of novel polysaccharide vaccines.
Assuntos
Células Apresentadoras de Antígenos/efeitos dos fármacos , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/farmacologia , Receptor 2 Toll-Like/agonistas , Células Apresentadoras de Antígenos/imunologia , Linhagem Celular , Humanos , Polissacarídeos Bacterianos/imunologiaRESUMO
Three plant xanthophylls are components of the xanthophyll cycle in which, upon exposure of leaves to high light, the enzyme violaxanthin de-epoxidase (VDE) transforms violaxanthin into zeaxanthin via the intermediate antheraxanthin. Previous work () showed that xanthophylls are bound to Lhc proteins and that substitution of violaxanthin with zeaxanthin induces conformational changes and fluorescence quenching by thermal dissipation. We have analyzed the efficiency of different Lhc proteins to exchange violaxanthin with zeaxanthin both in vivo and in vitro. Light stress of Zea mays leaves activates VDE, and the newly formed zeaxanthin is found primarily in CP26 and CP24, whereas other Lhc proteins show a lower exchange capacity. The de-epoxidation system has been reconstituted in vitro by using recombinant Lhc proteins, recombinant VDE, and monogalactosyl diacylglycerol (MGDG) to determine the intrinsic capacity for violaxanthin-to-zeaxanthin exchange of individual Lhc gene products. Again, CP26 was the most efficient in xanthophyll exchange. Biochemical and spectroscopic analysis of individual Lhc proteins after de-epoxidation in vitro showed that xanthophyll exchange occurs at the L2-binding site. Xanthophyll exchange depends on low pH, implying that access to the binding site is controlled by a conformational change via lumenal pH. These findings suggest that the xanthophyll cycle participates in a signal transduction system acting in the modulation of light harvesting versus thermal dissipation in the antenna system of higher plants.