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Marine organisms, including seagrasses, are important sources of biologically active molecules for the treatment of human diseases. In this study, organic extracts of the marine seagrass Halophila stipulacea obtained by different polarities from leaves (L) and stems (S) (hexane [HL, HS], ethyl acetate [EL, ES], and methanol [ML, MS]) were tested for different bioactivities. The screening comprehended the cytotoxicity activity against cancer cell lines grown as a monolayer culture or as multicellular spheroids (cancer), glucose uptake in cells (diabetes), reduction of lipid content in fatty acid-overloaded liver cells (steatosis), and lipid-reducing activity in zebrafish larvae (obesity), as well as the antifouling activity against marine bacteria (microfouling) and mussel larval settlement (macrofouling). HL, EL, HS, and ES extracts showed statistically significant cytotoxicity against cancer cell lines. The extracts did not have any significant effect on glucose uptake and on the reduction of lipids in liver cells. The EL and ML extracts reduced neutral lipid contents on the larvae of zebrafish with EC50 values of 2.2 µg/mL for EL and 1.2 µg/mL for ML. For the antifouling activity, the HS and ML extracts showed a significant inhibitory effect (p < 0.05) against the settlement of Mytilus galloprovincialis plantigrade larvae. The metabolite profiling using HR-LC-MS/MS and GNPS (The Global Natural Product Social Molecular Networking) analyses identified a variety of known primary and secondary metabolites in the extracts, along with some unreported molecules. Various compounds were detected with known activities on cancer (polyphenols: Luteolin, apeginin, matairesinol), on metabolic diseases (polyphenols: cirsimarin, spiraeoside, 2,4-dihydroxyheptadec-16-ynyl acetate; amino acids: N-acetyl-L-tyrosine), or on antifouling (fatty acids: 13-decosenamide; cinnamic acids: 3-hydroxy-4-methoxycinnamic acid, alpha-cyano-4-hydroxycinnamic), which could be, in part, responsible for the observed bioactivities. In summary, this study revealed that Halophila stipulacea is a rich source of metabolites with promising activities against obesity and biofouling and suggests that this seagrass could be useful for drug discovery in the future.
Assuntos
Antibacterianos/farmacologia , Hydrocharitaceae , Obesidade , Extratos Vegetais/farmacologia , Incrustação Biológica/prevenção & controle , Produtos BiológicosRESUMO
Marine organisms, particularly cyanobacteria, are important resources for the production of bioactive secondary metabolites for the treatment of human diseases. In this study, a bioassay-guided approach was used to discover metabolites with lipid-reducing activity. Two chlorophyll derivatives were successfully isolated, the previously described 132-hydroxy-pheophytin a (1) and the new compound 132-hydroxy-pheofarnesin a (2). The structure elucidation of the new compound 2 was established based on one- and two-dimensional (1D and 2D) NMR spectroscopy and mass spectrometry. Compounds 1 and 2 showed significant neutral lipid-reducing activity in the zebrafish Nile red fat metabolism assay after 48 h of exposure with a half maximal effective concentration (EC50) of 8.9 ± 0.4 µM for 1 and 15.5 ± 1.3 µM for 2. Both compounds additionally reduced neutral lipid accumulation in 3T3-L1 multicellular spheroids of murine preadipocytes. Molecular profiling of mRNA expression of some target genes was evaluated for the higher potent compound 1, which indicated altered peroxisome proliferator activated receptor gamma (PPARγ) mRNA expression. Lipolysis was not affected. Different food materials (Spirulina, Chlorella, spinach, and cabbage) were evaluated for the presence of 1, and the cyanobacterium Spirulina, with GRAS (generally regarded as safe) status for human consumption, contained high amounts of 1. In summary, known and novel chlorophyll derivatives were discovered from marine cyanobacteria with relevant lipid-reducing activities, which in the future may be developed into nutraceuticals.
Assuntos
Clorofila/análogos & derivados , Clorofila/farmacologia , Cianobactérias/química , Metabolismo dos Lipídeos/efeitos dos fármacos , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Brassica/química , Proteínas de Transporte/metabolismo , Linhagem Celular , Chlorella/química , Clorofila/química , Clorofila/isolamento & purificação , Ácido Graxo Sintase Tipo I/metabolismo , Lipólise , Camundongos , PPAR gama/metabolismo , Sirtuína 1/metabolismo , Spinacia oleracea/química , Spirulina/química , Peixe-ZebraRESUMO
Obesity is a complex disease resulting in several metabolic co-morbidities and is increasing at epidemic rates. The marine environment is an interesting resource of novel compounds and in particular cyanobacteria are well known for their capacity to produce novel secondary metabolites. In this work, we explored the potential of cyanobacteria for the production of compounds with relevant activities towards metabolic diseases using a blend of target-based, phenotypic and zebrafish assays as whole small animal models. A total of 46 cyanobacterial strains were grown and biomass fractionated, yielding in total 263 fractions. Bioactivities related to metabolic function were tested in different in vitro and in vivo models. Studying adipogenic and thermogenic gene expression in brown adipocytes, lipid metabolism and glucose uptake in hepatocytes, as well as lipid metabolism in zebrafish larvae, we identified 66 (25%) active fractions. This together with metabolite profiling and the evaluation of toxicity allowed the identification of 18 (7%) fractions with promising bioactivity towards different aspects of metabolic disease. Among those, we identified several known compounds, such as eryloside T, leptosin F, pheophorbide A, phaeophytin A, chlorophyll A, present as minor peaks. Those compounds were previously not described to have bioactivities in metabolic regulation, and both known or unknown compounds could be responsible for such effects. In summary, we find that cyanobacteria hold a huge repertoire of molecules with specific bioactivities towards metabolic diseases, which needs to be explored in the future.
Assuntos
Fármacos Antiobesidade/farmacologia , Cianobactérias/química , Obesidade/tratamento farmacológico , Adipócitos Marrons/efeitos dos fármacos , Adipócitos Marrons/fisiologia , Animais , Fármacos Antiobesidade/química , Fármacos Antiobesidade/toxicidade , Cianobactérias/crescimento & desenvolvimento , Cianobactérias/metabolismo , Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Células Hep G2 , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Obesidade/metabolismo , PPAR gama/metabolismo , Testes de Toxicidade , Proteína Desacopladora 1/metabolismo , Peixe-ZebraRESUMO
To increase the knowledge of the recombinant cyprosin production process in Saccharomyces cerevisiae cultures, it is relevant to implement efficient bioprocess monitoring techniques. The present work focuses on the implementation of a mid-infrared (MIR) spectroscopy-based tool for monitoring the recombinant culture in a rapid, economic, and high-throughput (using a microplate system) mode. Multivariate data analysis on the MIR spectra of culture samples was conducted. Principal component analysis (PCA) enabled capturing the general metabolic status of the yeast cells, as replicated samples appear grouped together in the score plot and groups of culture samples according to the main growth phase can be clearly distinguished. The PCA-loading vectors also revealed spectral regions, and the corresponding chemical functional groups and biomolecules that mostly contributed for the cell biomolecular fingerprint associated with the culture growth phase. These data were corroborated by the analysis of the samples' second derivative spectra. Partial least square (PLS) regression models built based on the MIR spectra showed high predictive ability for estimating the bioprocess critical variables: biomass (R 2 = 0.99, RMSEP 2.8%); cyprosin activity (R 2 = 0.98, RMSEP 3.9%); glucose (R 2 = 0.93, RMSECV 7.2%); galactose (R 2 = 0.97, RMSEP 4.6%); ethanol (R 2 = 0.97, RMSEP 5.3%); and acetate (R 2 = 0.95, RMSEP 7.0%). In conclusion, high-throughput MIR spectroscopy and multivariate data analysis were effective in identifying the main growth phases and specific cyprosin production phases along the yeast culture as well as in quantifying the critical variables of the process. This knowledge will promote future process optimization and control the recombinant cyprosin bioprocess according to Quality by Design framework.
Assuntos
Ácido Aspártico Endopeptidases/biossíntese , Proteínas Recombinantes/biossíntese , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Biomassa , Biotecnologia/métodos , Etanol/metabolismo , Galactose , Glucose/análise , Análise dos Mínimos Quadrados , Análise de Componente Principal , Análise de Regressão , Saccharomyces cerevisiae , Espectrofotometria Infravermelho , TemperaturaRESUMO
Reporter genes are routinely used in every laboratory for molecular and cellular biology for studying heterologous gene expression and general cellular biological mechanisms, such as transfection processes. Although well characterized and broadly implemented, reporter genes present serious limitations, either by involving time-consuming procedures or by presenting possible side effects on the expression of the heterologous gene or even in the general cellular metabolism. Fourier transform mid-infrared (FT-MIR) spectroscopy was evaluated to simultaneously analyze in a rapid (minutes) and high-throughput mode (using 96-wells microplates), the transfection efficiency, and the effect of the transfection process on the host cell biochemical composition and metabolism. Semi-adherent HEK and adherent AGS cell lines, transfected with the plasmid pVAX-GFP using Lipofectamine, were used as model systems. Good partial least squares (PLS) models were built to estimate the transfection efficiency, either considering each cell line independently (R (2) ≥ 0.92; RMSECV ≤ 2 %) or simultaneously considering both cell lines (R (2) = 0.90; RMSECV = 2 %). Additionally, the effect of the transfection process on the HEK cell biochemical and metabolic features could be evaluated directly from the FT-IR spectra. Due to the high sensitivity of the technique, it was also possible to discriminate the effect of the transfection process from the transfection reagent on KEK cells, e.g., by the analysis of spectral biomarkers and biochemical and metabolic features. The present results are far beyond what any reporter gene assay or other specific probe can offer for these purposes.
Assuntos
Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Transfecção , Genes Reporter , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Análise dos Mínimos Quadrados , MetabolomaRESUMO
An infant girl first presented with recurrent episodes of non-bilious vomiting, having had five hospitalisations over the following months because of dehydration. Laboratory data showed no inflammatory response, normal pancreatic amylase, but increased lipase levels (between 67 and 425 U/L). Several abdominal ultrasound studies suggested an intestinal duplication cyst on left hypochondrium and, later, a dilated and irregular pancreatic duct. CT showed a bifid tailed pancreas and a change in the cyst's characteristics. A communication with the pancreatic duct was hypothesised, which was confirmed on MR cholangiopancreatography. On laparoscopic surgery, the cyst was confirmed to be at the end of the caudal side of the pancreatic bifid tail, having no communication with the stomach. Cystectomy with partial pancreatectomy was performed with pathological findings confirming a gastric duplication cyst originating from the pancreatic bifid tail. At latest follow-up, 4 months after surgery, she remains asymptomatic.
Assuntos
Cistos , Anormalidades do Sistema Digestório , Lactente , Feminino , Humanos , Pâncreas/diagnóstico por imagem , Pâncreas/cirurgia , Ductos Pancreáticos , Pancreatectomia/efeitos adversos , Cistos/cirurgia , Anormalidades do Sistema Digestório/complicações , Vômito/etiologia , Vômito/cirurgiaRESUMO
We report the case of a pelvic malignant peripheral nerve sheath tumor mimicking an adnexal mass. A 59-year-old postmenopausal woman presented with a 3-month history of diffuse abdominal bloating and urinary frequency. Laboratory tests revealed an increased CA 125. Radiologic evaluation depicted a large, heterogeneous solid mass located right to the uterus, pushing it to the left. After a multidisciplinary board discussion, the diagnosis of a right adnexal lesion was assumed, and the patient was referred to surgery. The final diagnosis was only achieved after pathology examination, which prove to be a malignant peripheral nerve sheath tumor. This paper highlights some clinical, radiologic and pathological features of malignant peripheral nerve sheath tumors, a rare entity that should be considered as a differential in patients presenting with pelvic tumors of uncertain origin.
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Diffuse uterine leiomyomatosis is a rare and benign condition which involves the development of innumerable poorly defined, confluent smooth muscle nodules that replace most of the uterine parenchyma. It results in a symmetrically enlarged uterus. The etiology of these benign tumors is not completely understood. Patients with leiomyomatosis usually present with menorrhagia or dysmenorrhea, abdominal pain and infertility and in most cases hormonal treatment fails to control the symptoms. In this manuscript, we present a case of a 36-year-old woman who underwent hysterectomy due to diffuse uterine leiomyomatosis with a review of the literature.
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Mucoceles of the appendix are rare and can have quite variable imaging and clinical presentations, sometimes mimicking an adnexal mass. The underlying cause can be neoplastic or non-neoplastic. The typical imaging appearance of a mucocele of the appendix is that of a cystic structure with a tubular morphology. This structure is defined by having a blind-ending and being contiguous with the cecum. Radiologists should be familiar with key anatomical landmarks and with the various imaging features of mucoceles of the appendix, in order to provide a meaningful differential diagnosis of a lesion in the right lower abdominal quadrant. In addition, a neoplastic mucocele can rupture, resulting in pseudomyxoma peritonei, which will change the prognosis dramatically. Therefore, prompt diagnostic imaging is crucial.
Mucoceles do apêndice são raras e podem ter uma apresentação clínica e imagiológica bastante variável, por vezes mimetizando patologia anexial. As causas subjacentes podem ser neoplásicas ou não neoplásicas. O aspecto de imagem típico de mucoceles do apêndice é o de uma estrutura de natureza cística com morfologia tubular. Esta estrutura deverá terminar "em fundo cego" e ser contígua com o ceco. Os radiologistas devem estar familiarizados com os pontos anatômicos de referência e com as diferentes características imagiológicas de mucoceles do apêndice, de modo a fornecer um adequado diagnóstico diferencial de uma lesão localizada no quadrante abdominal inferior direito. Para além disso, uma mucocele neoplásica pode sofrer ruptura, resultando em pseudomixoma peritoneal, o que altera drasticamente o prognóstico. Assim, o diagnóstico por imagem em tempo útil é crucial.
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Microalgal biomass is widely studied for its possible application in food and human nutrition due to its multiple potential health benefits, and to address raising sustainability concerns. An interesting field whereby to further explore the application of microalgae is that of beer brewing, due to the capacity of some species to accumulate large amounts of starch under specific growth conditions. The marine species Tetraselmis chui is a well-known starch producer, and was selected in this study for the production of biomass to be explored as an active ingredient in beer brewing. Cultivation was performed under nitrogen deprivation in 250 L tubular photobioreactors, producing a biomass containing 50% starch. The properties of high-starch microalgal biomass in a traditional mashing process were then assessed to identify critical steps and challenges, test the efficiency of fermentable sugar release, and develop a protocol for small-scale brewing trials. Finally, T. chui was successfully integrated at a small scale into the brewing process as an active ingredient, producing microalgae-enriched beer containing up to 20% algal biomass. The addition of microalgae had a noticeable effect on the beer properties, resulting in a product with distinct sensory properties. Regulation of pH proved to be a key parameter in the process.
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Tetraselmis chui is known to accumulate starch when subjected to stress. This phenomenon is widely studied for the purpose of industrial production and process development. Yet, knowledge about the metabolic pathways involved is still immature. Hence, in this study, transcription of 27 starch-related genes was monitored under nitrogen deprivation and resupply in 25 L tubular photobioreactors. T. chui proved to be an efficient starch producer under nitrogen deprivation, accumulating starch up to 56% of relative biomass content. The prolonged absence of nitrogen led to an overall down-regulation of the tested genes, in most instances maintained even after nitrogen replenishment when starch was actively degraded. These gene expression patterns suggest post-transcriptional regulatory mechanisms play a key role in T. chui under nutrient stress. Finally, the high productivity combined with an efficient recovery after nitrogen restitution makes this species a suitable candidate for industrial production of high-starch biomass.
Assuntos
Clorófitas , Microalgas , Biomassa , Clorófitas/metabolismo , Redes e Vias Metabólicas , Microalgas/metabolismo , Nitrogênio/metabolismo , Amido/metabolismoRESUMO
INTRODUCTION: Dentinogenic ghost cell tumor (DGCT) is an entity with about 60 cases reported in the literature. It is a benign odontogenic tumor, despite being locally invasive and associated with a risk of local recurrence. PRESENTATION OF CASE: A 47-year-old woman presented with a 2-year-old expansive bone lesion. Radiologically, a multilocular mass was identified in the left superior maxilla, compatible with a tumor of odontogenic origin. She was submitted to an extended resection, and the histology was consistent with a DGCT. DISCUSSION: Central DGCT affects mainly male patients between the fourth and sixth decades, with a predilection for the posterior portion of the jaws. The symptoms are unspecific, and a vast percentage of patients is asymptomatic. Radiographically a unilocular feature is commonly found, unlike this case. The recommended treatment is extended local resection due to its high recurrence rate. CONCLUSION: Due to its rarity, knowledge of this entity is necessary for a better diagnostic and therapeutic guidance.
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Abstract Mucoceles of the appendix are rare and can have quite variable imaging and clinical presentations, sometimes mimicking an adnexal mass. The underlying cause can be neoplastic or non-neoplastic. The typical imaging appearance of a mucocele of the appendix is that of a cystic structure with a tubular morphology. This structure is defined by having a blind-ending and being contiguous with the cecum. Radiologists should be familiar with key anatomical landmarks and with the various imaging features of mucoceles of the appendix, in order to provide a meaningful differential diagnosis of a lesion in the right lower abdominal quadrant. In addition, a neoplastic mucocele can rupture, resulting in pseudomyxoma peritonei, which will change the prognosis dramatically. Therefore, prompt diagnostic imaging is crucial.
Resumo Mucoceles do apêndice são raras e podem ter uma apresentação clínica e imagiológica bastante variável, por vezes mimetizando patologia anexial. As causas subjacentes podem ser neoplásicas ou não neoplásicas. O aspecto de imagem típico de mucoceles do apêndice é o de uma estrutura de natureza cística com morfologia tubular. Esta estrutura deverá terminar "em fundo cego" e ser contígua com o ceco. Os radiologistas devem estar familiarizados com os pontos anatômicos de referência e com as diferentes características imagiológicas de mucoceles do apêndice, de modo a fornecer um adequado diagnóstico diferencial de uma lesão localizada no quadrante abdominal inferior direito. Para além disso, uma mucocele neoplásica pode sofrer ruptura, resultando em pseudomixoma peritoneal, o que altera drasticamente o prognóstico. Assim, o diagnóstico por imagem em tempo útil é crucial.
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Escherichia coli is one of the most used host microorganism for the production of recombinant products, such as heterologous proteins and plasmids. However, genetic, physiological and environmental factors influence the plasmid replication and cloned gene expression in a highly complex way. To control and optimize the recombinant expression system performance, it is very important to understand this complexity. Therefore, the development of rapid, highly sensitive and economic analytical methodologies, which enable the simultaneous characterization of the heterologous product synthesis and physiologic cell behavior under a variety of culture conditions, is highly desirable. For that, the metabolic profile of recombinant E. coli cultures producing the pVAX-lacZ plasmid model was analyzed by rapid, economic and high-throughput Fourier Transform Mid-Infrared (FT-MIR) spectroscopy. The main goal of the present work is to show as the simultaneous multivariate data analysis by principal component analysis (PCA) and direct spectral analysis could represent a very interesting tool to monitor E. coli culture processes and acquire relevant information according to current quality regulatory guidelines. While PCA allowed capturing the energetic metabolic state of the cell, e.g. by identifying different C-sources consumption phases, direct FT-MIR spectral analysis allowed obtaining valuable biochemical and metabolic information along the cell culture, e.g. lipids, RNA, protein synthesis and turnover metabolism. The information achieved by spectral multivariate data and direct spectral analyses complement each other and may contribute to understand the complex interrelationships between the recombinant cell metabolism and the bioprocess environment towards more economic and robust processes design according to Quality by Design framework. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:285-298, 2017.
Assuntos
Algoritmos , Escherichia coli/metabolismo , Perfilação da Expressão Gênica/métodos , Ensaios de Triagem em Larga Escala/métodos , Metaboloma/fisiologia , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Escherichia coli/genética , Análise Multivariada , Análise de Componente Principal , Recombinação Genética/genética , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Human mesenchymal stem/stromal cells (MSCs) have received considerable attention in the field of cell-based therapies due to their high differentiation potential and ability to modulate immune responses. However, since these cells can only be isolated in very low quantities, successful realization of these therapies requires MSCs ex-vivo expansion to achieve relevant cell doses. The metabolic activity is one of the parameters often monitored during MSCs cultivation by using expensive multi-analytical methods, some of them time-consuming. The present work evaluates the use of mid-infrared (MIR) spectroscopy, through rapid and economic high-throughput analyses associated to multivariate data analysis, to monitor three different MSCs cultivation runs conducted in spinner flasks, under xeno-free culture conditions, which differ in the type of microcarriers used and the culture feeding strategy applied. After evaluating diverse spectral preprocessing techniques, the optimized partial least square (PLS) regression models based on the MIR spectra to estimate the glucose, lactate and ammonia concentrations yielded high coefficients of determination (R(2) ≥ 0.98, ≥0.98, and ≥0.94, respectively) and low prediction errors (RMSECV ≤ 4.7%, ≤4.4% and ≤5.7%, respectively). Besides PLS models valid for specific expansion protocols, a robust model simultaneously valid for the three processes was also built for predicting glucose, lactate and ammonia, yielding a R(2) of 0.95, 0.97 and 0.86, and a RMSECV of 0.33, 0.57, and 0.09 mM, respectively. Therefore, MIR spectroscopy combined with multivariate data analysis represents a promising tool for both optimization and control of MSCs expansion processes. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:447-455, 2016.
Assuntos
Reatores Biológicos , Técnicas de Cultura de Células , Células-Tronco Mesenquimais/citologia , Ensaios de Triagem em Larga Escala , Humanos , Análise Multivariada , Espectroscopia de Luz Próxima ao InfravermelhoRESUMO
The development of biopharmaceutical manufacturing processes presents critical constraints, with the major constraint being that living cells synthesize these molecules, presenting inherent behavior variability due to their high sensitivity to small fluctuations in the cultivation environment. To speed up the development process and to control this critical manufacturing step, it is relevant to develop high-throughput and in situ monitoring techniques, respectively. Here, high-throughput mid-infrared (MIR) spectral analysis of dehydrated cell pellets and in situ near-infrared (NIR) spectral analysis of the whole culture broth were compared to monitor plasmid production in recombinant Escherichia coli cultures. Good partial least squares (PLS) regression models were built, either based on MIR or NIR spectral data, yielding high coefficients of determination (R(2)) and low predictive errors (root mean square error, or RMSE) to estimate host cell growth, plasmid production, carbon source consumption (glucose and glycerol), and by-product acetate production and consumption. The predictive errors for biomass, plasmid, glucose, glycerol, and acetate based on MIR data were 0.7 g/L, 9 mg/L, 0.3 g/L, 0.4 g/L, and 0.4 g/L, respectively, whereas for NIR data the predictive errors obtained were 0.4 g/L, 8 mg/L, 0.3 g/L, 0.2 g/L, and 0.4 g/L, respectively. The models obtained are robust as they are valid for cultivations conducted with different media compositions and with different cultivation strategies (batch and fed-batch). Besides being conducted in situ with a sterilized fiber optic probe, NIR spectroscopy allows building PLS models for estimating plasmid, glucose, and acetate that are as accurate as those obtained from the high-throughput MIR setup, and better models for estimating biomass and glycerol, yielding a decrease in 57 and 50% of the RMSE, respectively, compared to the MIR setup. However, MIR spectroscopy could be a valid alternative in the case of optimization protocols, due to possible space constraints or high costs associated with the use of multi-fiber optic probes for multi-bioreactors. In this case, MIR could be conducted in a high-throughput manner, analyzing hundreds of culture samples in a rapid and automatic mode.
Assuntos
Reatores Biológicos , Biotecnologia , Proteínas Recombinantes/metabolismo , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Biotecnologia/métodos , Biotecnologia/normas , Meios de Cultura/química , Escherichia coli/metabolismo , Análise dos Mínimos QuadradosRESUMO
Near infrared (NIR) spectroscopy was used to in situ monitoring the cultivation of two recombinant Saccharomyces cerevisiae strains producing heterologous cyprosin B. NIR spectroscopy is a fast and non-destructive technique, that by being based on overtones and combinations of molecular vibrations requires chemometrics tools, such as partial least squares (PLS) regression models, to extract quantitative information concerning the variables of interest from the spectral data. In the present work, good PLS calibration models based on specific regions of the NIR spectral data were built for estimating the critical variables of the cyprosin production process: biomass concentration, cyprosin activity, cyprosin specific activity, the carbon sources glucose and galactose concentration and the by-products acetic acid and ethanol concentration. The PLS models developed are valid for both recombinant S. cerevisiae strains, presenting distinct cyprosin production capacities, and therefore can be used, not only for the real-time control of both processes, but also in optimization protocols. The PLS model for biomass yielded a R(2)=0.98 and a RMSEP=0.46 g dcw l(-1), representing an error of 4% for a calibration range between 0.44 and 13.75 g dcw l(-1). A R(2)=0.94 and a RMSEP=167 Um l(-1) were obtained for the cyprosin activity, corresponding to an error of 6.7% of the experimental data range (0-2509 Um l(-1)), whereas a R(2)=0.93 and RMSEP=672 U mg(-1) were obtained for the cyprosin specific activity, corresponding to an error of 7% of the experimental data range (0-11,690 Um g(-1)). For the carbon sources glucose and galactose, a R(2)=0.96 and a RMSECV of 1.26 and 0.55 g l(-1), respectively, were obtained, showing high predictive capabilities within the range of 0-20 g l(-1). For the metabolites resulting from the cell growth, the PLS model for acetate was characterized by a R(2)=0.92 and a RMSEP=0.06 g l (-1), which corresponds to a 6.1% error within the range of 0.41-1.23 g l(-1); for the ethanol, a high accuracy PLS model with a R(2)=0.97 and a RMSEP=1.08 g l(-1) was obtained, representing an error of 9% within the range of 0.18-21.76 g l(-1). The present study shows that it is possible the in situ monitoring and prediction of the critical variables of the recombinant cyprosin B production process by NIR spectroscopy, which can be applied in process control in real-time and in optimization protocols. From the above, NIR spectroscopy appears as a valuable analytical tool for online monitoring of cultivation processes, in a fast, accurate and reproducible operation mode.
Assuntos
Ácido Aspártico Endopeptidases/biossíntese , Recombinação Genética , Saccharomyces cerevisiae/metabolismo , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Biomassa , Saccharomyces cerevisiae/genéticaRESUMO
A finalidade deste estudo foi descrever o uso da monitorização com base no currículo (MBC) - fluência, na identificação de alunos em risco de apresentarem dislexia. Participaram 146 alunos do 3º ano do 1º Ciclo do Ensino Básico dum agrupamento de escolas do concelho de Braga, Portugal, que foram monitorizados em dois momentos ao longo do ano letivo. Foram considerados alunos em risco, aqueles cujo resultado se encontrava abaixo ou no percentil 20. Os resultados permitiram concluir que: 1) A prova de MBC mostrou ser econômica, fácil e rápida de aplicar, de cotar e bem aceita pelos professores e alunos; 2) Em média, a prestação dos alunos na primeira aplicação foi de 85,21 (DP = 28,41) palavras corretas por minuto (pcpm), na segunda aplicação (final do ano letivo) foi de 97,46 (DP = 30,07) pcpm; 3) O crescimento semanal foi de 0,49 (DP=0,38) pcpm; 4) Encontram-se em risco de apresentarem dislexia, após a segunda aplicação 11 rapazes e 18 raparigas; 5) A diferença de resultados entre turmas é estatisticamente significativa; 6) No final do ano letivo em duas turmas mais de 30% dos alunos estão em risco de apresentarem dislexia, tendo em conta o valor do risco da amostra; 7) 70,55% dos participantes não atingiram o objetivo das 110 pcpm estipulado pelo Ministério da Educação para aquele ano de escolaridade; 8) O valor do Alfa de Cronbach para a primeira aplicação foi de 0,981 e para a segunda aplicação foi de 0,978.
The purpose of this study was to describe the use of curriculum-based monitoring (CBM) of reading fluency for identifying students at risk for presenting dyslexia. One hundred and forty six third grade students from a group of schools in Braga, Portugal, participated in the study; they were monitored twice during the school year. The students whose result was below or in the 20th percentile were considered at risk. The results show that: 1) The CBM reading fluency test was economic, quick and easy to apply and measure and well accepted by teachers and students; 2) On average, the students' results for the first application were 85.21 (SD=28.41) correct words per minute (cwpm), and 97.46 (SD=30.07) cwpm in the second application (at the end of the school year); 3) The weekly increase was 0.49 (SD=0.38) cwpm; 4) After the second application, 11 boys and 18 girls were considered at risk for presenting dyslexia; 5) The difference in results between class groups was statistically significant; 6) Considering the sample results, at the end of the school year in two classes, more than 30% of students were at risk; 7) 70.55 % did not reach the goal of 110 cwpm stipulated by the Ministry of Education for that school level; 8) The Cronbach's Alpha coefficient for the first application was 0.981 and 0.978 for the second application.