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2.
Artigo em Inglês | MEDLINE | ID: mdl-34580764

RESUMO

Despite the conventional view that a truly random V(D)J recombination process should generate a highly diverse immune repertoire, emerging reports suggest that there is a certain bias toward the generation of shared/public immune receptor chains. These studies were performed in viral diseases where public T cell receptors (TCR) appear to confer better protective responses. Selective pressures generating common TCR clonotypes are currently not well understood, but it is believed that they confer a growth advantage. As very little is known about public TCR clonotypes in cancer, here we set out to determine the extent of shared TCR clonotypes in the intra-tumor microenvironments of virus- and non-virus-driven head and neck cancers using TCR sequencing. We report that tumor-infiltrating T cell clonotypes were indeed shared across individuals with the same cancer type, where the majority of shared sequences were specific to the cancer type (i.e., viral versus non-viral). These shared clonotypes were not particularly enriched in EBV-associated nasopharynx cancer but, in both cancers, exhibited distinct characteristics, namely shorter CDR3 lengths, restricted V- and J-gene usages, and also demonstrated convergent V(D)J recombination. Many of these shared TCRs were expressed in patients with a shared HLA background. Pattern recognition of CDR3 amino acid sequences revealed strong convergence to specific pattern motifs, and these motifs were uniquely found to each cancer type. This suggests that they may be enriched for specificity to common antigens found in the tumor microenvironment of different cancers. The identification of shared TCRs in infiltrating tumor T cells not only adds to our understanding of the tumor-adaptive immune recognition but could also serve as disease-specific biomarkers and guide the development of future immunotherapies.

3.
Nature ; 593(7860): 597-601, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33902106

RESUMO

N6-methyladenosine (m6A) is an abundant internal RNA modification1,2 that is catalysed predominantly by the METTL3-METTL14 methyltransferase complex3,4. The m6A methyltransferase METTL3 has been linked to the initiation and maintenance of acute myeloid leukaemia (AML), but the potential of therapeutic applications targeting this enzyme remains unknown5-7. Here we present the identification and characterization of STM2457, a highly potent and selective first-in-class catalytic inhibitor of METTL3, and a crystal structure of STM2457 in complex with METTL3-METTL14. Treatment of tumours with STM2457 leads to reduced AML growth and an increase in differentiation and apoptosis. These cellular effects are accompanied by selective reduction of m6A levels on known leukaemogenic mRNAs and a decrease in their expression consistent with a translational defect. We demonstrate that pharmacological inhibition of METTL3 in vivo leads to impaired engraftment and prolonged survival in various mouse models of AML, specifically targeting key stem cell subpopulations of AML. Collectively, these results reveal the inhibition of METTL3 as a potential therapeutic strategy against AML, and provide proof of concept that the targeting of RNA-modifying enzymes represents a promising avenue for anticancer therapy.

4.
Nat Cancer ; 1(2): 249-263, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32118208

RESUMO

Mutational signatures are patterns of mutations that arise during tumorigenesis. We present an enhanced, practical framework for mutational signature analyses. Applying these methods on 3,107 whole genome sequenced (WGS) primary cancers of 21 organs reveals known signatures and nine previously undescribed rearrangement signatures. We highlight inter-organ variability of signatures and present a way of visualizing that diversity, reinforcing our findings in an independent analysis of 3,096 WGS metastatic cancers. Signatures with a high level of genomic instability are dependent on TP53 dysregulation. We illustrate how uncertainty in mutational signature identification and assignment to samples affects tumor classification, reinforcing that using multiple orthogonal mutational signature data is not only beneficial, it is essential for accurate tumor stratification. Finally, we present a reference web-based tool for cancer and experimentally-generated mutational signatures, called Signal (https://signal.mutationalsignatures.com), that also supports performing mutational signature analyses.

5.
Nat Commun ; 10(1): 4543, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31586074

RESUMO

Sequencing studies of diffuse large B cell lymphoma (DLBCL) have identified hundreds of recurrently altered genes. However, it remains largely unknown whether and how these mutations may contribute to lymphomagenesis, either individually or in combination. Existing strategies to address this problem predominantly utilize cell lines, which are limited by their initial characteristics and subsequent adaptions to prolonged in vitro culture. Here, we describe a co-culture system that enables the ex vivo expansion and viral transduction of primary human germinal center B cells. Incorporation of CRISPR/Cas9 technology enables high-throughput functional interrogation of genes recurrently mutated in DLBCL. Using a backbone of BCL2 with either BCL6 or MYC, we identify co-operating genetic alterations that promote growth or even full transformation into synthetically engineered DLBCL models. The resulting tumors can be expanded and sequentially transplanted in vivo, providing a scalable platform to test putative cancer genes and to create mutation-directed, bespoke lymphoma models.


Assuntos
Linfócitos B/patologia , Linfoma Difuso de Grandes Células B/genética , Cultura Primária de Células/métodos , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proliferação de Células/genética , Técnicas de Cocultura/métodos , Vetores Genéticos/genética , Centro Germinativo/citologia , Ensaios de Triagem em Larga Escala , Humanos , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Gradação de Tumores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas Proto-Oncogênicas c-myc/genética , Retroviridae/genética , Transdução Genética , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Artigo em Inglês | MEDLINE | ID: mdl-31645345

RESUMO

"Mutational signatures" are patterns of mutations that report DNA damage and subsequent repair processes that have occurred. Whole-genome sequencing (WGS) can provide additional information to standard diagnostic techniques and can identify therapeutic targets. A 32-yr-old male with xeroderma pigmentosum developed metastatic angiosarcoma that was unresponsive to three lines of conventional sarcoma therapies. WGS was performed on his primary cancer revealing a hypermutated tumor, including clonal ultraviolet radiation-induced mutational patterns (Signature 7) and subclonal signatures of mutated DNA polymerase epsilon (POLE) (Signature 10). These signatures are associated with response to immune checkpoint blockade. Immunohistochemistry confirmed high PD-L1 expression in metastatic deposits. The anti-PD-1 monoclonal antibody pembrolizumab was commenced off-label given the POLE mutation and high mutational load. After four cycles, there was a significant reduction in his disease with almost complete resolution of the metastatic deposits. This case highlights the importance of WGS in the analysis, interpretation, and treatment of cancers. We anticipate that as WGS becomes integral to the cancer diagnostic pathway, treatments will be stratified to the individual based on their unique genomic and/or transcriptomic profile, enhancing classical approaches of histologically driven treatment decisions.


Assuntos
Hemangiossarcoma/genética , Xeroderma Pigmentoso/tratamento farmacológico , Xeroderma Pigmentoso/genética , Adulto , Anticorpos Monoclonais/genética , Anticorpos Monoclonais Humanizados/uso terapêutico , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/genética , Análise Mutacional de DNA/métodos , DNA Polimerase II/genética , Humanos , Masculino , Instabilidade de Microssatélites , Mutação/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genética , Sequenciamento Completo do Genoma/métodos
7.
Anal Bioanal Chem ; 411(20): 5115-5126, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31152220

RESUMO

Despite technological advances, two-dimensional electrophoresis (2DE) of biological fluids, such as vitreous, remains a major challenge. In this study, artificial neural network was applied to optimize the recovery of vitreous proteins and its detection by 2DE analysis through the combination of several solubilizing agents (CHAPS, Genapol, DTT, IPG buffer), temperature, and total voltage. The highest protein recovery (94.9% ± 4.5) was achieved using 4% (w/v) CHAPS, 0.1% (v/v) Genapol, 20 mM DTT, and 2% (v/v) IPG buffer. Two iterations were required to achieve an optimized response (580 spots) using 4% (w/v) CHAPS, 0.2% (v/v) Genapol, 60 mM DTT, and 0.5% (v/v) IPG buffer at 35 kVh and 25 °C, representing a 2.4-fold improvement over the standard initial conditions of the experimental design. The analysis of depleted vitreous using the optimized protocol resulted in an additional 1.3-fold increment in protein detection over the optimal output, with an average of 761 spots detected in vitreous from different vitreoretinopathies. Our results clearly indicate the importance of combining the appropriate amount of solubilizing agents with a suitable control of the temperature and voltage to obtain high-quality gels. The high-throughput of this model provides an effective starting point for the optimization of 2DE protocols. This experimental design can be adapted to other types of matrices. Graphical abstract.


Assuntos
Eletroforese em Gel Bidimensional/métodos , Redes Neurais de Computação , Proteômica/métodos , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
8.
Nat Commun ; 9(1): 5378, 2018 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-30568163

RESUMO

We recently identified the splicing kinase gene SRPK1 as a genetic vulnerability of acute myeloid leukemia (AML). Here, we show that genetic or pharmacological inhibition of SRPK1 leads to cell cycle arrest, leukemic cell differentiation and prolonged survival of mice transplanted with MLL-rearranged AML. RNA-seq analysis demonstrates that SRPK1 inhibition leads to altered isoform levels of many genes including several with established roles in leukemogenesis such as MYB, BRD4 and MED24. We focus on BRD4 as its main isoforms have distinct molecular properties and find that SRPK1 inhibition produces a significant switch from the short to the long isoform at the mRNA and protein levels. This was associated with BRD4 eviction from genomic loci involved in leukemogenesis including BCL2 and MYC. We go on to show that this switch mediates at least part of the anti-leukemic effects of SRPK1 inhibition. Our findings reveal that SRPK1 represents a plausible new therapeutic target against AML.


Assuntos
Leucemia Mieloide Aguda/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular , Diferenciação Celular , Cromatina/metabolismo , Epigênese Genética , Células HL-60 , Hematopoese , Humanos , Células K562 , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Splicing de RNA
9.
Blood ; 131(15): 1639-1653, 2018 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-29463564

RESUMO

FLT3 internal tandem duplication (FLT3ITD) mutations are common in acute myeloid leukemia (AML) associated with poor patient prognosis. Although new-generation FLT3 tyrosine kinase inhibitors (TKI) have shown promising results, the outcome of FLT3ITD AML patients remains poor and demands the identification of novel, specific, and validated therapeutic targets for this highly aggressive AML subtype. Utilizing an unbiased genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screen, we identify GLS, the first enzyme in glutamine metabolism, as synthetically lethal with FLT3-TKI treatment. Using complementary metabolomic and gene-expression analysis, we demonstrate that glutamine metabolism, through its ability to support both mitochondrial function and cellular redox metabolism, becomes a metabolic dependency of FLT3ITD AML, specifically unmasked by FLT3-TKI treatment. We extend these findings to AML subtypes driven by other tyrosine kinase (TK) activating mutations and validate the role of GLS as a clinically actionable therapeutic target in both primary AML and in vivo models. Our work highlights the role of metabolic adaptations as a resistance mechanism to several TKI and suggests glutaminolysis as a therapeutically targetable vulnerability when combined with specific TKI in FLT3ITD and other TK activating mutation-driven leukemias.


Assuntos
Glutamina/metabolismo , Leucemia Mieloide Aguda , Mutação , Inibidores de Proteínas Quinases/farmacologia , Tirosina Quinase 3 Semelhante a fms , Sistemas CRISPR-Cas , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Estudo de Associação Genômica Ampla , Glutamina/genética , Humanos , Células K562 , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Células THP-1 , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo
10.
Microbiology (Reading) ; 163(6): 829-839, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28635591

RESUMO

Multiple interacting factors affect the performance of engineered biological systems in synthetic biology projects. The complexity of these biological systems means that experimental design should often be treated as a multiparametric optimization problem. However, the available methodologies are either impractical, due to a combinatorial explosion in the number of experiments to be performed, or are inaccessible to most experimentalists due to the lack of publicly available, user-friendly software. Although evolutionary algorithms may be employed as alternative approaches to optimize experimental design, the lack of simple-to-use software again restricts their use to specialist practitioners. In addition, the lack of subsidiary approaches to further investigate critical factors and their interactions prevents the full analysis and exploitation of the biotechnological system. We have addressed these problems and, here, provide a simple-to-use and freely available graphical user interface to empower a broad range of experimental biologists to employ complex evolutionary algorithms to optimize their experimental designs. Our approach exploits a Genetic Algorithm to discover the subspace containing the optimal combination of parameters, and Symbolic Regression to construct a model to evaluate the sensitivity of the experiment to each parameter under investigation. We demonstrate the utility of this method using an example in which the culture conditions for the microbial production of a bioactive human protein are optimized. CamOptimus is available through: (https://doi.org/10.17863/CAM.10257).


Assuntos
Biologia Computacional/métodos , Muramidase/biossíntese , Pichia/genética , Algoritmos , Evolução Biológica , Biotecnologia , Biologia Computacional/instrumentação , Humanos , Internet , Muramidase/genética , Pichia/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Software
12.
Blood ; 128(1): e1-9, 2016 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-27121471

RESUMO

The diagnosis of hematologic malignancies relies on multidisciplinary workflows involving morphology, flow cytometry, cytogenetic, and molecular genetic analyses. Advances in cancer genomics have identified numerous recurrent mutations with clear prognostic and/or therapeutic significance to different cancers. In myeloid malignancies, there is a clinical imperative to test for such mutations in mainstream diagnosis; however, progress toward this has been slow and piecemeal. Here we describe Karyogene, an integrated targeted resequencing/analytical platform that detects nucleotide substitutions, insertions/deletions, chromosomal translocations, copy number abnormalities, and zygosity changes in a single assay. We validate the approach against 62 acute myeloid leukemia, 50 myelodysplastic syndrome, and 40 blood DNA samples from individuals without evidence of clonal blood disorders. We demonstrate robust detection of sequence changes in 49 genes, including difficult-to-detect mutations such as FLT3 internal-tandem and mixed-lineage leukemia (MLL) partial-tandem duplications, and clinically significant chromosomal rearrangements including MLL translocations to known and unknown partners, identifying the novel fusion gene MLL-DIAPH2 in the process. Additionally, we identify most significant chromosomal gains and losses, and several copy neutral loss-of-heterozygosity mutations at a genome-wide level, including previously unreported changes such as homozygosity for DNMT3A R882 mutations. Karyogene represents a dependable genomic diagnosis platform for translational research and for the clinical management of myeloid malignancies, which can be readily adapted for use in other cancers.


Assuntos
Genômica/métodos , Neoplasias Hematológicas , Leucemia Mieloide , Síndromes Mielodisplásicas , Proteínas de Transporte/genética , DNA (Citosina-5-)-Metiltransferases/genética , Feminino , Forminas , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/genética , Histona-Lisina N-Metiltransferase/genética , Humanos , Leucemia Mieloide/diagnóstico , Leucemia Mieloide/genética , Masculino , Mutação , Síndromes Mielodisplásicas/diagnóstico , Síndromes Mielodisplásicas/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Fusão Oncogênica/genética , Tirosina Quinase 3 Semelhante a fms/genética
13.
Microb Cell Fact ; 14: 113, 2015 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-26246150

RESUMO

BACKGROUND: Membrane proteins are important drug targets in many human diseases and gathering structural information regarding these proteins encourages the pharmaceutical industry to develop new molecules using structure-based drug design studies. Specifically, membrane-bound catechol-O-methyltransferase (MBCOMT) is an integral membrane protein that catalyzes the methylation of catechol substrates and has been linked to several diseases such as Parkinson's disease and Schizophrenia. Thereby, improvements in the clinical outcome of the therapy to these diseases may come from structure-based drug design where reaching MBCOMT samples in milligram quantities are crucial for acquiring structural information regarding this target protein. Therefore, the main aim of this work was to optimize the temperature, dimethylsulfoxide (DMSO) concentration and the methanol flow-rate for the biosynthesis of recombinant MBCOMT by Pichia pastoris bioreactor methanol-induced cultures using artificial neural networks (ANN). RESULTS: The optimization trials intended to evaluate MBCOMT expression by P. pastoris bioreactor cultures led to the development of a first standard strategy for MBCOMT bioreactor biosynthesis with a batch growth on glycerol until the dissolved oxygen spike, 3 h of glycerol feeding and 12 h of methanol induction. The ANN modeling of the aforementioned fermentation parameters predicted a maximum MBCOMT specific activity of 384.8 nmol/h/mg of protein at 30°C, 2.9 mL/L/H methanol constant flow-rate and with the addition of 6% (v/v) DMSO with almost 90% of healthy cells at the end of the induction phase. These results allowed an improvement of MBCOMT specific activity of 6.4-fold in comparison to that from the small-scale biosynthesis in baffled shake-flasks. CONCLUSIONS: The ANN model was able to describe the effects of temperature, DMSO concentration and methanol flow-rate on MBCOMT specific activity, as shown by the good fitness between predicted and observed values. This experimental procedure highlights the potential role of chemical chaperones such as DMSO in improving yields of recombinant membrane proteins with a different topology than G-coupled receptors. Finally, the proposed ANN shows that the manipulation of classic fermentation parameters coupled with the addition of specific molecules can open and reinforce new perspectives in the optimization of P. pastoris bioprocesses for membrane proteins biosynthesis.


Assuntos
Catecol O-Metiltransferase/biossíntese , Membrana Celular/enzimologia , Meios de Cultura/química , Metanol/metabolismo , Pichia/metabolismo , Reatores Biológicos/microbiologia , Catecol O-Metiltransferase/química , Catecol O-Metiltransferase/genética , Catecóis/metabolismo , Membrana Celular/genética , Meios de Cultura/metabolismo , Fermentação , Humanos , Redes Neurais de Computação , Pichia/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Temperatura
14.
N Biotechnol ; 31(4): 335-44, 2014 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-23933561

RESUMO

In this work, we present a dynamic metabolic model that describes the uptake of complex mixtures of volatile fatty acids (VFA) and respective conversion into PHA by mixed microbial cultures (MMC). This model builds upon a previously published flux balance analysis model [1] that identified the minimization of TCA cycle activity as the key metabolic objective to predict PHA storage fluxes and respective composition. The model was calibrated either with experimental data of PHA production from fermented sugar cane molasses or from synthetic mixtures of VFA. All PHA production experiments were performed using a MMC selected with fermented sugar cane molasses under feast and famine regimen. The model was able to capture the process dynamics denoted by an excellent fit between experimental and computed time profiles of concentrations with the regression coefficients always above 0.92. The introduced VFA uptake regulatory factor reflects the decrease of acetyl-CoA and propionyl-CoA available to TCA cycle in conformity with the hypothesis that the minimization of TCA cycle is a key metabolic objective for MMC subjected to feast and famine regimen for the maximization of PHA production.


Assuntos
Bactérias/metabolismo , Reatores Biológicos/microbiologia , Ácidos Graxos Voláteis/metabolismo , Modelos Teóricos , Poli-Hidroxialcanoatos/metabolismo , Técnicas de Cultura Celular por Lotes , Simulação por Computador , Fermentação , Melaço , Saccharum/metabolismo
15.
Microb Cell Fact ; 12: 51, 2013 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-23692918

RESUMO

BACKGROUND: Novel analytical tools, which shorten the long and costly development cycles of biopharmaceuticals are essential. Metabolic flux analysis (MFA) shows great promise in improving our understanding of the metabolism of cell factories in bioreactors, but currently only provides information post-process using conventional off-line methods. MFA combined with real time multianalyte process monitoring techniques provides a valuable platform technology allowing real time insights into metabolic responses of cell factories in bioreactors. This could have a major impact in the bioprocessing industry, ultimately improving product consistency, productivity and shortening development cycles. RESULTS: This is the first investigation using Near Infrared Spectroscopy (NIRS) in situ combined with metabolic flux modelling which is both a significant challenge and considerable extension of these techniques. We investigated the feasibility of our approach using the industrial workhorse Pichia pastoris in a simplified model system. A parental P. pastoris strain (i.e. which does not synthesize recombinant protein) was used to allow definition of distinct metabolic states focusing solely upon the prediction of intracellular fluxes in central carbon metabolism. Extracellular fluxes were determined using off-line conventional reference methods and on-line NIR predictions (calculated by multivariate analysis using the partial least squares algorithm, PLS). The results showed that the PLS-NIRS models for biomass and glycerol were accurate: correlation coefficients, R2, above 0.90 and the root mean square error of prediction, RMSEP, of 1.17 and 2.90 g/L, respectively. The analytical quality of the NIR models was demonstrated by direct comparison with the standard error of the laboratory (SEL), which showed that performance of the NIR models was suitable for quantifying biomass and glycerol for calculating extracellular metabolite rates and used as independent inputs for the MFA (RMSEP lower than 1.5 × SEL). Furthermore, the results for the MFA from both datasets passed consistency tests performed for each steady state, showing that the precision of on-line NIRS is equivalent to that obtained by the off-line measurements. CONCLUSIONS: The findings of this study show for the first time the potential of NIRS as an input generating for MFA models, contributing to the optimization of cell factory metabolism in real-time.


Assuntos
Pichia/metabolismo , Algoritmos , Biomassa , Glicerol/metabolismo , Análise do Fluxo Metabólico , Modelos Biológicos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Espectroscopia de Luz Próxima ao Infravermelho
16.
Water Sci Technol ; 67(5): 1008-16, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23416592

RESUMO

Phenomenological models and hybrid phenomenological-chemometric models were developed to predict natural organic matter (NOM) removal based on the real water treatment data from the city of Minneapolis over a 3 year period. The analysis of the modeling results showed that the phenomenological model was able to capture the major variations of NOM removal but it tended to over predict the NOM removal in independent data sets. These results could be significantly improved by the hybrid model, which was less biased and much more accurate than the phenomenological model. The phenomenological model parameters showed low statistical confidence because the available data, collected in real water treatment conditions, was not sufficiently informative to identify the complex model structure. By comparison, the hybrid modeling method enabled a more reliable discrimination of the most important factors affecting NOM removal. The final hybrid model was implemented in an Excel spreadsheet and can be easily used for NOM removal prediction and the control of chemical dosing.


Assuntos
Modelos Teóricos , Compostos Orgânicos/isolamento & purificação , Poluentes Químicos da Água/isolamento & purificação , Propriedades de Superfície
17.
Adv Biochem Eng Biotechnol ; 132: 193-215, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23272320

RESUMO

In this chapter we explore the basic tools for the design of bioprocess monitoring, optimization, and control algorithms that incorporate a priori knowledge of metabolic networks. The main advantage is that this ultimately enables the targeting of intracellular control variables such as metabolic reactions or metabolic pathways directly linked with productivity and product quality. We analyze in particular design methods that target elementary modes of metabolic networks. The topics covered include the analysis of the structure of metabolic networks, computation and reduction of elementary modes, measurement methods for the envirome, envirome-guided metabolic reconstruction, and macroscopic dynamic modeling and control. These topics are illustrated with applications to a cultivation process of a recombinant Pichia pastoris X33 strain expressing a single-chain antibody fragment (scFv).


Assuntos
Redes e Vias Metabólicas/fisiologia , Transdução de Sinais/fisiologia , Algoritmos , Biologia Computacional/métodos , Modelos Biológicos , Pichia/metabolismo , Pichia/fisiologia , Anticorpos de Cadeia Única/metabolismo
18.
J Biotechnol ; 162(2-3): 336-45, 2012 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-23036926

RESUMO

Fermented agro-industrial wastes are potential low cost substrates for polyhydroxyalkanoates (PHA) production by mixed microbial cultures (MMC). The use of complex substrates has however profound implications in the PHA metabolism. In this paper we investigate PHA accumulation using a lumped metabolic model that describes PHA storage from arbitrary mixtures of volatile fatty acids (VFA). Experiments were conducted using synthetic and complex VFA mixtures obtained from the fermentation of sugar cane molasses. Metabolic flux analysis (MFA) and flux balance analysis (FBA) were performed at different stages of culture enrichment in order to investigate the effect of VFA composition and time of enrichment in PHA storage efficiency. Substrate uptake and PHA storage fluxes increased over enrichment time by 70% and 73%, respectively. MFA calculations show that higher PHA storage fluxes are associated to an increase in the uptake of VFA with even number of carbon atoms and a more effective synthesis of hydroxyvalerate (HV) precursors from VFA with odd number of carbons. Furthermore, FBA shows that the key metabolic objective of a MMC subjected to the feast and famine regimen is the minimization of the tricarboxylic acid cycle fluxes. The PHA flux and biopolymer composition (hydroxybutyrate (HB): HV) could be accurately predicted in several independent experiments.


Assuntos
Reatores Biológicos/microbiologia , Ácidos Graxos Voláteis/metabolismo , Modelos Biológicos , Poli-Hidroxialcanoatos/metabolismo , Biotecnologia , Fermentação , Cinética , Redes e Vias Metabólicas , Melaço , Saccharum , Biologia de Sistemas/métodos
19.
BMC Syst Biol ; 5: 181, 2011 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-22044634

RESUMO

BACKGROUND: Elementary flux modes (EFM) are unique and non-decomposable sets of metabolic reactions able to operate coherently in steady-state. A metabolic network has in general a very high number of EFM reflecting the typical functional redundancy of biological systems. However, most of these EFM are either thermodynamically unfeasible or inactive at pre-set environmental conditions. RESULTS: Here we present a new algorithm that discriminates the "active" set of EFM on the basis of dynamic envirome data. The algorithm merges together two well-known methods: projection to latent structures (PLS) and EFM analysis, and is therefore termed projection to latent pathways (PLP). PLP has two concomitant goals: (1) maximisation of correlation between EFM weighting factors and measured envirome data and (2) minimisation of redundancy by eliminating EFM with low correlation with the envirome. CONCLUSIONS: Overall, our results demonstrate that PLP slightly outperforms PLS in terms of predictive power. But more importantly, PLP is able to discriminate the subset of EFM with highest correlation with the envirome, thus providing in-depth knowledge of how the environment controls core cellular functions. This offers a significant advantage over PLS since its abstract structure cannot be associated with the underlying biological structure.


Assuntos
Algoritmos , Redes e Vias Metabólicas , Modelos Biológicos , Animais , Linhagem Celular , Cricetinae , Biologia de Sistemas , Termodinâmica
20.
Biotechnol Bioeng ; 102(4): 1098-106, 2009 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-18853411

RESUMO

The main objective of the present study was to investigate the use of in situ 2D fluorometry for monitoring key bioprocess variables in mammalian cell cultures, namely the concentration of viable cells and the concentration of recombinant proteins. All studies were conducted using a recombinant Baby Hamster Kidney (BHK) cell line expressing a fusion glycoprotein IgG1-IL2 cultured in batch and fed-batch modes. It was observed that the intensity of fluorescence signals in the excitation/emission wavelength range of amino acids, vitamins and NAD(P)H changed along culture time, although the dynamics of single fluorophors could not be correlated with the dynamics of the target state variables. Therefore, multivariate chemometric modeling was adopted as a calibration methodology. 2D fluorometry produced large volumes of redundant spectral data, which were first filtered by principal components analysis (PCA). Then, a partial least squares (PLS) regression was applied to correlate the reduced fluorescence maps with the target state variables. Two validation strategies were used to evaluate the predictive capacity of the developed PLS models. Accurate estimations of viable cells density (r(2) = 0.95; 99.2% of variance captured in the training set; r(2) = 0.91; 97.7% of variance captured in the validation set) and of glycoprotein concentration (r(2) = 0.99 and 99.7% of variance captured in the training set; r(2) = 0.99 and 99.3% of variance captured in the validation set) were obtained over a wide range of reactor operation conditions. The results presented herein confirm that 2D fluorometry constitutes a reliable methodology for on-line monitoring of viable cells and recombinant protein concentrations in mammalian cell cultures.


Assuntos
Técnicas de Cultura de Células/métodos , Fluorometria/métodos , Proteínas Recombinantes de Fusão/biossíntese , Animais , Reatores Biológicos , Linhagem Celular , Sobrevivência Celular , Cricetinae , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , Interleucina-2/biossíntese , Interleucina-2/genética , Proteínas Recombinantes de Fusão/genética , Reprodutibilidade dos Testes
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