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1.
Nucleic Acids Res ; 51(18): 10059-10074, 2023 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-37678882

RESUMO

Implementation of therapeutic in vivo gene editing using CRISPR/Cas relies on potent delivery of gene editing tools. Administration of ribonucleoprotein (RNP) complexes consisting of Cas protein and single guide RNA (sgRNA) offers short-lived editing activity and safety advantages over conventional viral and non-viral gene and RNA delivery approaches. By engineering lentivirus-derived nanoparticles (LVNPs) to facilitate RNP delivery, we demonstrate effective administration of SpCas9 as well as SpCas9-derived base and prime editors (BE/PE) leading to gene editing in recipient cells. Unique Gag/GagPol protein fusion strategies facilitate RNP packaging in LVNPs, and refinement of LVNP stoichiometry supports optimized LVNP yield and incorporation of therapeutic payload. We demonstrate near instantaneous target DNA cleavage and complete RNP turnover within 4 days. As a result, LVNPs provide high on-target DNA cleavage and lower levels of off-target cleavage activity compared to standard RNP nucleofection in cultured cells. LVNPs accommodate BE/sgRNA and PE/epegRNA RNPs leading to base editing with reduced bystander editing and prime editing without detectable indel formation. Notably, in the mouse eye, we provide the first proof-of-concept for LVNP-directed in vivo gene disruption. Our findings establish LVNPs as promising vehicles for delivery of RNPs facilitating donor-free base and prime editing without formation of double-stranded DNA breaks.

2.
Br J Haematol ; 202(4): 825-839, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37190875

RESUMO

The frontline therapy R-CHOP for patients with diffuse large B-cell lymphoma (DLBCL) has remained unchanged for two decades despite numerous Phase III clinical trials investigating new alternatives. Multiple large studies have uncovered genetic subtypes of DLBCL enabling a targeted approach. To further pave the way for precision oncology, we perform genome-wide CRISPR screening to uncover the cellular response to one of the components of R-CHOP, vincristine, in the DLBCL cell line SU-DHL-5. We discover important pathways and subnetworks using gene-set enrichment analysis and protein-protein interaction networks and identify genes related to mitotic spindle organization that are essential during vincristine treatment. The inhibition of KIF18A, a mediator of chromosome alignment, using the small molecule inhibitor BTB-1 causes complete cell death in a synergistic manner when administered together with vincristine. We also identify the genes KIF18B and USP28 of which CRISPR/Cas9-directed knockout induces vincristine resistance across two DLBCL cell lines. Mechanistic studies show that lack of KIF18B or USP28 counteracts a vincristine-induced p53 response suggesting that resistance to vincristine has origin in the mitotic surveillance pathway (USP28-53BP1-p53). Collectively, our CRISPR screening data uncover potential drug targets and mechanisms behind vincristine resistance, which may support the development of future drug regimens.


Assuntos
Linfoma Difuso de Grandes Células B , Proteína Supressora de Tumor p53 , Humanos , Vincristina/farmacologia , Vincristina/uso terapêutico , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Medicina de Precisão , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Rituximab/uso terapêutico , Pontos de Checagem do Ciclo Celular , Apoptose , Ciclofosfamida/uso terapêutico , Prednisona/uso terapêutico , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ubiquitina Tiolesterase , Cinesinas/genética
3.
Phys Rev Lett ; 126(23): 230404, 2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34170163

RESUMO

We observe monopole oscillations in a mixture of Bose-Einstein condensates, where the usually dominant mean-field interactions are canceled. In this case, the system is governed by the next-order Lee-Huang-Yang (LHY) correction to the ground state energy, which describes the effect of quantum fluctuations. Experimentally such a LHY fluid is realized by controlling the atom numbers and interaction strengths in a ^{39}K spin mixture confined in a spherical trap potential. We measure the monopole oscillation frequency as a function of the LHY interaction strength as proposed recently by Jrgensen et al. [Phys. Rev. Lett. 121, 173403 (2018)PRLTAO0031-900710.1103/PhysRevLett.121.173403] and find excellent agreement with simulations of the complete experiment including the excitation procedure and inelastic losses. This confirms that the system and its collective behavior are initially dominated by LHY interactions. Moreover, the monopole oscillation frequency is found to be stable against variations of the involved scattering lengths in a broad region around the ideal values, confirming the stabilizing effect of the LHY interaction. These results pave the way for using the nonlinearity provided by the LHY term in quantum simulation experiments and for investigations beyond the LHY regime.

4.
Acta Paediatr ; 108(5): 842-848, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-29926969

RESUMO

AIM: Necrotising enterocolitis (NEC) is often staged according to Bell's 1978 system, but today's NEC cases are more immature than the ones that were used to develop Bell's stages. Our aim was to explore the clinical and radiographic findings of contemporary cases of NEC and spontaneous intestinal perforation. METHODS: We coded the clinical records of all cases of NEC stages I-III and spontaneous intestinal perforation born in 2006-2015 at the tertiary department of neonatology at Rigshospitalet, Denmark, for 16 clinical and radiographic symptoms and signs at disease onset and at climax. These variables were explored using principal component analysis, which can detect patterns in large datasets. RESULTS: We reviewed 640 clinical records and included 158 cases of NEC or spontaneous intestinal perforation. When we entered the clinical and radiographic signs at disease climax, the cases were roughly grouped according to Bell's stages, except for a small group of NEC III cases, who were grouped with the cases of spontaneous intestinal perforation. CONCLUSION: An analysis of the pattern of clinical and radiographic findings in a 2006-2015 population of NEC cases supported Bell's 1978 staging system. However, the separation between NEC and spontaneous intestinal perforation still poses a difficult task.


Assuntos
Enterocolite Necrosante/diagnóstico , Doenças do Prematuro/diagnóstico , Perfuração Intestinal/diagnóstico , Dinamarca , Feminino , Humanos , Recém-Nascido , Masculino , Análise de Componente Principal , Radiografia , Estudos Retrospectivos , Índice de Gravidade de Doença
5.
Anal Bioanal Chem ; 408(1): 83-96, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26573172

RESUMO

Lactic acid bacteria with antifungal properties are applied for biopreservation of food. In order to further our understanding of their antifungal mechanism, there is an ongoing search for bioactive molecules. With a focus on the metabolites formed, bioassay-guided fractionation and comprehensive screening have identified compounds as antifungal. Although these are active, the compounds have been found in concentrations that are too low to account for the observed antifungal effect. It has been hypothesized that the formation of metabolites and consumption of nutrients during bacterial fermentations form the basis for the antifungal effect, i.e., the composition of the exometabolome. To build a more comprehensive view of the chemical changes induced by bacterial fermentation and the effects on mold growth, a strategy for correlating the exometabolomic profiles with mold growth was applied. The antifungal properties were assessed by measuring mold growth of two Penicillium strains on cell-free ferments of three strains of Lactobacillus paracasei pre-fermented in a chemically defined medium. Exometabolomic profiling was performed by reversed-phase liquid chromatography in combination with mass spectrometry in electrospray positive and negative modes. By multivariate data analysis, the three strains of Lb. paracasei were readily distinguished by the relative difference of their exometabolomes. The relative differences correlated with the relative growth of the two Penicillium strains. Metabolic footprinting proved to be a supplement to bioassay-guided fractionation for investigation of antifungal properties of bacterial ferments. Additionally, three previously identified and three novel antifungal metabolites from Lb. paracasei and their potential precursors were detected and assigned using the strategy.


Assuntos
Antifúngicos/metabolismo , Antifúngicos/farmacologia , Lactobacillus/metabolismo , Antifúngicos/química , Cromatografia de Fase Reversa , Lactobacillus/química , Espectrometria de Massas , Metabolômica , Penicillium/efeitos dos fármacos , Penicillium/crescimento & desenvolvimento
6.
Anal Chem ; 86(15): 7160-70, 2014 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-24978618

RESUMO

We demonstrate how to process comprehensive two-dimensional gas chromatograms (GC × GC chromatograms) to remove nonsample information (artifacts), including background and retention time shifts. We also demonstrate how this, combined with further reduction of the influence of irrelevant information, allows for data analysis without integration or peak deconvolution (pixel-based analysis).

7.
Appl Environ Microbiol ; 80(9): 2889-900, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24584251

RESUMO

Fecal samples were obtained from a cohort of 330 healthy Danish infants at 9, 18, and 36 months after birth, enabling characterization of interbacterial relationships by use of quantitative PCR targeting 31 selected bacterial 16S rRNA gene targets representing different phylogenetic levels. Nutritional parameters and measures of growth and body composition were determined and investigated in relation to the observed development in microbiota composition. We found that significant changes in the gut microbiota occurred, particularly from age 9 to 18 months, when cessation of breastfeeding and introduction of a complementary feeding induce replacement of a microbiota characterized by lactobacilli, bifidobacteria, and Enterobacteriaceae with a microbiota dominated by Clostridium spp. and Bacteroides spp. Classification of samples by a proxy enterotype based on the relative levels of Bacteroides spp. and Prevotella spp. showed that enterotype establishment occurs between 9 and 36 months. Thirty percent of the individuals shifted enterotype between 18 and 36 months. The composition of the microbiota was most pronouncedly influenced by the time of cessation of breastfeeding. From 9 to 18 months, a positive correlation was observed between the increase in body mass index and the increase of the short-chain-fatty-acid-producing clostridia, the Clostridum leptum group, and Eubacterium hallii. Considering previously established positive associations between rapid infant weight gain, early breastfeeding discontinuation, and later-life obesity, the corresponding microbial findings seen here warrant attention.


Assuntos
Bactérias/isolamento & purificação , Intestinos/microbiologia , Microbiota , Bactérias/classificação , Bactérias/genética , Aleitamento Materno , Pré-Escolar , Estudos de Coortes , DNA Bacteriano/genética , Dinamarca , Fezes/microbiologia , Feminino , Humanos , Lactente , Masculino , RNA Ribossômico 16S/genética
8.
Anaerobe ; 28: 68-77, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24905430

RESUMO

Prebiotic oligosaccharides are defined by their selective stimulation of growth and/or activity of bacteria in the digestive system in ways claimed to be beneficial for health. However, apart from the short chain fatty acids, little is known about bacterial metabolites created by fermentation of prebiotics, and the significance of the size of the oligosaccharides remains largely unstudied. By in vitro fermentations in human fecal microbial communities (derived from six different individuals), we studied the effects of high-mass (HA, >1 kDa), low-mass (LA, <1 kDa) and mixed (BA) sugar beet arabino-oligosaccharides (AOS) as carbohydrate sources. Fructo-oligosaccharides (FOS) were included as reference. The changes in bacterial communities and the metabolites produced in response to incubation with the different carbohydrates were analyzed by quantitative PCR (qPCR) and Liquid Chromatography-Mass Spectrometry (LC-MS), respectively. All tested carbohydrate sources resulted in a significant increase of Bifidobacterium spp. between 1.79 fold (HA) and 1.64 fold (FOS) in the microbial populations after fermentation, and LC-MS analysis suggested that the bifidobacteria contributed to decomposition of the arabino-oligosaccharide structures, most pronounced in the HA fraction, resulting in release of the essential amino acid phenylalanine. Abundance of Lactobacillus spp. correlated with the presence of a compound, most likely a flavonoid, indicating that lactobacilli contribute to release of such health-promoting substances from plant structures. Additionally, the combination of qPCR and LC-MS revealed a number of other putative interactions between intestinal microbes and the oligosaccharides, which contributes to the understanding of the mechanisms behind prebiotic impact on human health.


Assuntos
Bactérias/efeitos dos fármacos , Trato Gastrointestinal/microbiologia , Metaboloma , Microbiota/efeitos dos fármacos , Oligossacarídeos/metabolismo , Filogenia , Prebióticos , Adulto , Bactérias/genética , Bactérias/metabolismo , Cromatografia Líquida , Feminino , Fermentação , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Peso Molecular , Oligossacarídeos/química , Reação em Cadeia da Polimerase em Tempo Real
9.
Analyst ; 138(12): 3502-11, 2013 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-23665697

RESUMO

An automated method (FastChrom) for baseline correction, peak detection and assignment (grouping) of similar peaks across samples has been developed. The method has been tested both on artificial data and a dataset obtained from gas chromatograph analysis of wine samples. As part of the automated approach, a new method for baseline estimation has been developed and compared with other methods. FastChrom has been shown to perform at least as well as conventional software. However, compared to other approaches, FastChrom finds more peaks in the chromatograms and not only those with retention times defined by the user. FastChrom is fast and easy to use and offers the possibility of applying a retention time index which facilitates the identification of peaks and the comparison between experiments.


Assuntos
Cromatografia/métodos , Estatística como Assunto/métodos , Automação , Modelos Teóricos
10.
Anal Bioanal Chem ; 405(25): 8151-70, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23954995

RESUMO

Co-cultures of specific lactic and propionic acid bacteria have been shown to have an antagonistic effect against yeast and moulds in dairy systems. In studies of these co-cultures by bioassay-guided fractionation and analysis, numerous compounds have been reported to inhibit yeast and moulds. Although active, the compounds do not account for the full effect observed. Instead, the inhibitory action in the co-culture is believed to be a result of synergy between known exo-metabolites, depletion of nutrients, and/or compounds not yet identified. Untargeted metabolomics or metabolic footprinting could be a potent approach to elucidation of the mechanism. The purpose of this review is to discuss the two pre-requisites for such a study--the compound classes expected in the co-cultures, and on the basis of these, the most suitable analytical technique(s). Ultrahigh-performance liquid chromatography (UPLC) coupled to high-resolution mass spectrometry (MS) via electrospray ionisation (ESI) operated in both positive and negative modes is regarded as the optimum instrumental technique. The applicability of a range of liquid chromatographic techniques ranging from ion-pair (IPC) and hydrophilic interaction (HILIC) to reversed-phase chromatography (RPC) is discussed in terms of the expected metabolome. Use of both HILIC and RPC is suggested, on account of the complementarity of these modes. The most promising strategy uses a combination of the two electrospray polarities and two modes of LC. The strategy recommended in this study does not include all compound classes, and suggestions for supplementary methods are listed.


Assuntos
Bactérias/metabolismo , Cromatografia Líquida/métodos , Ácido Láctico/análise , Espectrometria de Massas/métodos , Metabolômica/métodos , Propionatos/análise , Bactérias/química , Cromatografia Líquida/instrumentação , Cromatografia de Fase Reversa/instrumentação , Cromatografia de Fase Reversa/métodos , Técnicas de Cocultura , Ácido Láctico/metabolismo , Espectrometria de Massas/instrumentação , Metabolômica/instrumentação , Propionatos/metabolismo
11.
J Sci Food Agric ; 93(15): 3710-9, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23633436

RESUMO

BACKGROUND: Visible-near infrared spectroscopy remains a method of increasing interest as a fast alternative for the evaluation of fruit quality. The success of the method is assumed to be achieved by using large sets of samples to produce robust calibration models. In this study we used representative samples of an early and a late season apple cultivar to evaluate model robustness (in terms of prediction ability and error) on the soluble solids content (SSC) and acidity prediction, in the wavelength range 400-1100 nm. RESULTS: A total of 196 middle-early season and 219 late season apples (Malus domestica Borkh.) cvs 'Aroma' and 'Holsteiner Cox' samples were used to construct spectral models for SSC and acidity. Partial least squares (PLS), ridge regression (RR) and elastic net (EN) models were used to build prediction models. Furthermore, we compared three sub-sample arrangements for forming training and test sets ('smooth fractionator', by date of measurement after harvest and random). Using the 'smooth fractionator' sampling method, fewer spectral bands (26) and elastic net resulted in improved performance for SSC models of 'Aroma' apples, with a coefficient of variation CVSSC = 13%. The model showed consistently low errors and bias (PLS/EN: R(2) cal = 0.60/0.60; SEC = 0.88/0.88°Brix; Biascal = 0.00/0.00; R(2) val = 0.33/0.44; SEP = 1.14/1.03; Biasval = 0.04/0.03). However, the prediction acidity and for SSC (CV = 5%) of the late cultivar 'Holsteiner Cox' produced inferior results as compared with 'Aroma'. CONCLUSION: It was possible to construct local SSC and acidity calibration models for early season apple cultivars with CVs of SSC and acidity around 10%. The overall model performance of these data sets also depend on the proper selection of training and test sets. The 'smooth fractionator' protocol provided an objective method for obtaining training and test sets that capture the existing variability of the fruit samples for construction of visible-NIR prediction models. The implication is that by using such 'efficient' sampling methods for obtaining an initial sample of fruit that represents the variability of the population and for sub-sampling to form training and test sets it should be possible to use relatively small sample sizes to develop spectral predictions of fruit quality. Using feature selection and elastic net appears to improve the SSC model performance in terms of R(2), RMSECV and RMSEP for 'Aroma' apples.


Assuntos
Ácidos/análise , Calibragem , Frutas/química , Malus/química , Modelos Biológicos , Estações do Ano , Ingestão de Alimentos , Frutas/normas , Humanos , Malus/classificação , Reprodutibilidade dos Testes , Solubilidade , Especificidade da Espécie , Espectroscopia de Luz Próxima ao Infravermelho/métodos
12.
Pediatr Res ; 71(1): 115-20, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22289859

RESUMO

INTRODUCTION: The pathophysiology of necrotizing enterocolitis (NEC) is multifactorial, and gastrointestinal bacteria are thought to play an important role. In this study, the role of microflora in the gastrointestinal tract of neonates with NEC was assessed by comparing cases with controls. RESULTS: Of the 163 neonates, 21 developed NEC. The risk of NEC decreased by 8% with each additional day of gestational age. DISCUSSION: Typically, very few bacterial species could be cultured from the fecal specimens obtained. Gram-positive (G(+)) bacteria dominated the samples in the NEC group, whereas in the control group mixed flora of G(+) and Gram-negative (G(-)) bacteria were isolated. Surprisingly, molecular analysis using PCR-DGGE profiles did not confirm these differences. Our data suggest that G(+) bacteria in the intestine may play a role in the development of NEC in premature infants. METHODS: One hundred and sixty three neonates born at <30 weeks of gestation were enrolled. Fecal samples taken during the first month of life were subjected to culture and PCR-denaturing gradient gel electrophoresis (PCR-DGGE). A total of 482 fecal samples were examined.


Assuntos
Enterocolite Necrosante/microbiologia , Enterocolite Necrosante/patologia , Doenças do Prematuro/microbiologia , Doenças do Prematuro/patologia , Intestinos/microbiologia , Intestinos/patologia , Enterobacteriaceae/isolamento & purificação , Enterocolite Necrosante/fisiopatologia , Fezes/microbiologia , Feminino , Idade Gestacional , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Doenças do Prematuro/fisiopatologia , Intestinos/fisiopatologia , Masculino , Análise de Componente Principal
13.
J Allergy Clin Immunol ; 128(3): 646-52.e1-5, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21782228

RESUMO

BACKGROUND: Changes in the human microbiome have been suggested as a risk factor for a number of lifestyle-related disorders, such as atopic diseases, possibly through a modifying influence on immune maturation in infancy. OBJECTIVES: We aimed to explore the association between neonatal fecal flora and the development of atopic disorders until age 6 years, hypothesizing that the diversity of the intestinal microbiota influences disease development. METHODS: We studied the intestinal microbiota in infants in the Copenhagen Prospective Study on Asthma in Childhood, a clinical study of a birth cohort of 411 high-risk children followed for 6 years by clinical assessments at 6-month intervals, as well as at acute symptom exacerbations. Bacterial flora was analyzed at 1 and 12 months of age by using molecular techniques based on 16S rRNA PCR combined with denaturing gradient gel electrophoresis, as well as conventional culturing. The main outcome measures were the development of allergic sensitization (skin test and specific serum IgE), allergic rhinitis, peripheral blood eosinophil counts, asthma, and atopic dermatitis during the first 6 years of life. RESULTS: We found that bacterial diversity in the early intestinal flora 1 and 12 months after birth was inversely associated with the risk of allergic sensitization (serum specific IgE P = .003; skin prick test P = .017), peripheral blood eosinophils (P = .034), and allergic rhinitis (P = .007). There was no association with the development of asthma or atopic dermatitis. CONCLUSIONS: Reduced bacterial diversity of the infant's intestinal flora was associated with increased risk of allergic sensitization, allergic rhinitis, and peripheral blood eosinophilia, but not asthma or atopic dermatitis, in the first 6 years of life. These results support the general hypothesis that an imbalance in the intestinal microbiome is influencing the development of lifestyle-related disorders, such as allergic disease.


Assuntos
Bactérias/classificação , Hipersensibilidade Imediata/epidemiologia , Intestinos/microbiologia , Metagenoma , Bactérias/genética , Bactérias/isolamento & purificação , Criança , Estudos de Coortes , DNA Bacteriano/análise , Eletroforese em Gel de Gradiente Desnaturante , Eosinofilia/diagnóstico , Eosinofilia/epidemiologia , Fezes/microbiologia , Humanos , Hipersensibilidade Imediata/diagnóstico , Imunoglobulina E/sangue , Lactente , Recém-Nascido , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , Rinite/diagnóstico , Rinite/epidemiologia , Risco , Testes Cutâneos
14.
Mol Ther Nucleic Acids ; 29: 563-576, 2022 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-36090759

RESUMO

Locus-directed DNA cleavage induced by the CRISPR-Cas9 system triggers DNA repair mechanisms allowing gene repair or targeted insertion of foreign DNA. For gene insertion to be successful, availability of a homologous donor template needs to be timed with cleavage of the DNA by the Cas9 endonuclease guided by a target-specific single guide RNA (sgRNA). We present a novel approach for targeted gene insertion based on a single integrase-defective lentiviral vector (IDLV) carrying a Cas9 off switch. Gene insertion using this approach benefits from transposon-based stable Cas9 expression, which is switched off by excision-only transposase protein co-delivered in IDLV particles carrying a combined sgRNA/donor vector. This one-vector approach supports potent (up to >80%) knockin of a full-length EGFP gene sequence. This traceless cell engineering method benefits from high stable levels of Cas9, timed intracellular availability of the molecular tools, and a built-in feature to turn off Cas9 expression after DNA cleavage. The simple technique is based on transduction with a single IDLV, which holds the capacity to transfer larger donor templates, allowing robust gene knockin or tagging of genes in a single step.

15.
N Biotechnol ; 56: 54-62, 2020 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-31770609

RESUMO

Real-time monitoring of bioprocesses plays a key-role in modern industries, providing new information on full-scale production, thus enabling control of the process and allowing it to run at optimal conditions while minimizing waste. Monitoring of phosphates and ammonium in fermentation processes has a twofold interest: they are important nutrients for living organisms while at the same time constituting environmental nutrient pollutants, for which unnecessary use and disposal must be avoided. In this report, the possibility of simultaneous analysis of phosphates and ammonium in fermentations was verified using spectroscopy-based methods combined with chemometrics to construct calibration models. To achieve this, the models were based on synthetic samples mimicking real fermentation media, providing a dataset where the analytes were completely uncorrelated. Different at-line techniques (mid- and near- infrared spectroscopy, MIR and NIR) were evaluated for their ability to monitor quickly both analytes, in a wide range of concentrations (10-100 mM), in three media of different complexities. Partial Least Squares (PLS) models on MIR spectroscopy gave very good results, with prediction errors lower than 5 % for both analytes in all datasets. In contrast, the results for PLS models on NIR spectroscopy were inferior (prediction errors between 3 and 26 %) for both analytes, as, in the case of phosphate, it could be demonstrated that the model was based on based on indirect predictions.


Assuntos
Compostos de Amônio/análise , Fermentação , Fosfatos/análise , Compostos de Amônio/metabolismo , Calibragem , Estudos de Viabilidade , Análise dos Mínimos Quadrados , Fosfatos/metabolismo , Espectroscopia de Luz Próxima ao Infravermelho
16.
Appl Spectrosc ; 73(9): 1087-1098, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31008650

RESUMO

The ever-growing competition among global biotech industries has led to high demands on production consistency. A statistical strategy of performance mapping for production optimization is therefore of great economic significance. Process analytical technology (PAT)-based sensors such as mid-infrared (MIR) spectroscopy enable process monitoring through substrate and by-product concentrations that directly represent the physiology of cells. Combined with multivariate statistics, MIR can be employed as a strategy for production performance mapping. This study describes the use of at-line spectroscopy, chemometric modeling, and post-process fitting to characterize Lactobacillus acidophilus fermentations. The emphasis is on alternative arrangements of the data and chemometric methods principle component analysis (PCA), multivariate curve resolution (MCR), and parallel factor analysis (PARAFAC). Two key parameters, rate constant and time of inflection, are extracted by post-process fitting on the outcomes of these different models. Their use as process performance descriptors to characterize the dynamics of substrate consumption, product formation and batch-to-batch variations is suggested. The unconstrained PCA primarily described biomass change, while the constrained models PARAFAC and MCR (both the augmented and individual-run configurations) could model the decrease in sugars and increase in lactic acid over time. It was concluded that MCR on individual batch data, followed by post-process fitting, is the preferred strategy for MIR spectroscopic monitoring.


Assuntos
Biotecnologia/métodos , Meios de Cultura/metabolismo , Fermentação , Lactobacillus acidophilus/crescimento & desenvolvimento , Análise de Componente Principal/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Lactobacillus acidophilus/metabolismo , Análise Multivariada
17.
Biotechnol Prog ; 35(2): e2762, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30507037

RESUMO

Different opportunities are explored to evaluate quality variation in raw materials from biological origin. Assessment of raw materials attributes is an important step in a bio-based production as fluctuations in quality are a major source of process disturbance. This can be due to a variety of biological, seasonal, and supply scarcity reasons. The final properties of a product are invariably linked with the initial properties of the raw material. Thus, the operational conditions of a process can be tuned to drive the product to the required specification based on the quality assessment of the raw material being processed. Process analytical technology tools which enable this assessment in a far more informative and rapid manner than current industrial practices that rely on rule-of-thumb decisions are assessed. An example with citrus peels is used to demonstrate the conceptual and performance differences of distinct quality assessment approaches. The analysis demonstrates the advantage of characterization through multivariate data analysis coupled with a complementary spectroscopic technique, near-infrared spectroscopy. The quantitative comparative analysis of three different approaches, discriminant classification based on expert-knowledge, unsupervised classification, and spectroscopic correlation with reference physicochemical variables, is performed in the same dataset context. © 2018 Her Majesty the Queen in Right of Canada © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2762, 2019.


Assuntos
Produtos Biológicos/análise , Pectinas/análise , Análise Multivariada , Espectroscopia de Luz Próxima ao Infravermelho
18.
Biotechnol Prog ; 34(1): 231-242, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29063712

RESUMO

In this study, the aim was to establish if loss of DNA integrity is a cause of loss of culturability for probiotic bacteria during storage in dry state. The number of colony forming units (CFU), number of metabolically active cells, and DNA integrity during dry storage of probiotic strains, B. animalis subsp. lactis BB-12 and L. acidophilus LA-5, were investigated. The probiotic strains were freeze-dried and stored at 20°C, with and without oxygen present, and at water activity levels 0.22 or 0.32. Dry storage resulted in a decrease in CFU during the entire storage period. The number of metabolically active cells was unchanged during storage of B. animalis subsp. lactis BB-12, but did decrease during the first week of storage of L. acidophilus LA-5. Loss of DNA integrity was evident for both strains during storage and correlated well with the loss of CFU. Both loss of CFU and loss of DNA integrity were significantly greater for both strains when oxygen was present and when aw was increased. Statistical analysis indicates a possible causal relationship between DNA degradation and loss of culturability and this idea is consistent with the function of DNA at cell division. The study contributes with new knowledge of the cause for loss of CFU during dry storage of probiotic bacteria, which possibly can aid in the improvement of preservation techniques. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:231-242, 2018.


Assuntos
Fragmentação do DNA , Genoma Bacteriano/genética , Genômica , Probióticos , Bifidobacterium/genética , Bifidobacterium/crescimento & desenvolvimento , Liofilização , Lactobacillus acidophilus/genética , Lactobacillus acidophilus/crescimento & desenvolvimento , Células-Tronco/metabolismo
20.
Food Res Int ; 102: 256-264, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29195947

RESUMO

Protein hydrolysates are of great interest in the food industry due to their nutritional and functional properties, but their use often implies solubilization in water and therefore hamper the use of plant proteins with inherent low water solubility. Protein solubility in water can be modified by enzymatic hydrolysis, but during this process several collateral properties of the protein hydrolysates changes. It is therefore important to determine the end-point of the process and to monitor its development. In this feasibility study, we demonstrated the potential of different spectroscopic techniques (1H NMR and IR) coupled with chemometrics analysis in monitoring the hydrolysis of five different industrial grade plant proteins by the enzyme Alcalase. Logarithmic modeling of the PCA (Principal Component Analysis) scores confirmed that they can represent a measurement of the solubilized protein material released and resulted in kinetic parameters describing the suitability of protein sources as substrates for the hydrolysis. This way, we showed that a qualitative evaluation of the degree of hydrolysis is possible using fast at-line technologies and PCA.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Proteínas de Plantas/metabolismo , Subtilisinas/metabolismo , Estudos de Viabilidade , Hidrólise , Proteínas de Plantas/química , Solubilidade , Espectrofotometria Infravermelho , Vibração
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