Extraction and spectrophotometric and fluorimetric determination of micrograms of zinc in plants

9 pág.[ES] Se han aplicado dos nuevos métodos originales, espectrofotométrico y fluorimétrico, a la determinación de microgramos de cinc en material vegetal. Después de la disolución de la muestra, el cinc se extrae en solución 0,05 M de trioctilamina en tolueno, desde soluciones 1-3 M HCl en fase acuosa. El desarrollo del color o de la fluorescencia se efectua en la misma fase orgánica de la extracción sin efectuar re-extracción a fase acuosa, mediante adición de solución de oxina en N,N-dimetilformamida (DMF) y de solución de t-butil-amina en DMF. La absorbancia se mide a 403 nm. La ley de Beer se cumple en el intervalo de 1 a 10 mg. ml-1 de cinc en la fase orgánica final. La desviación estandar relativa para diez determinaciones de 50 mg. de cinc fue de 0,8%. En el método fluorimétrico, se ha empleado una radiación de excitación de 403 nm de longitud de onda y una emisión de fluorescencia de 450 nm. La intensidad relativa de fluorescencia de 450 nm. La intensidad relativa de fluorescencia era lineal con la concentración de cinc en el intervalo de 0,01 a 1 mg. ml-1) en la fase orgánica final. La desviación estandar relativa en la determinación fluorimétrica para diez determinaciones de 0,5 mg. de cinc fué de 2,2%. El valor medio de la recuperación de 0,5 mg de cinc añadidos a las soluciones de las muestras fué del 98,5%. Los resultados obtenidos en la determinación de cinc por ambos métodos propuestos en muestras de material vegetal suministradas amablemente por M. PINTA del Comité Inter-Institutos de Análisis Foliar, muestran buena precisión y exactitud.[EN] Two new spectrophotometric and fluorimetric methods for micrograms determination of zinc have been applied to plants analysis. After dissolution of the sample, zinc was extracted into 0.05 M trioctylamine in toluene solution from 1-3 M HCl aqueous solution. The development of the colour or the fluorescence was carried out in the same organic phase of the extraction, without back-extraction into aqueous phase, by addition of oxine solution in N, N-dimenthylformamide and t-butylamine in DMF solution. In the spectrophotometric method the absorbance was measured at 403 nm. Beer's law was obeyed in the range from 1 up to 10µg. ml-1 of zinc in the organic phase. The relative standard deviation for ten determinations of 50 µg. of zinc was 0,8%. In the f1uorimetric method a wavelength excitation radiation of 403 nm and a wavelength fluorescence emission of 450 nm were used. The relative intensity of f1uorescence was linear with the concentration of zinc in the range from 0.01 up to 1 µg. ml-1 of zinc in the organic phase. The standard relative deviation in the f1uorimetric determination for ten determinations of 0.5 µg of zinc was 2.2%. The mean recovery of 0.5 µg of zinc in spiked solutions was 98.5%. The results obtained for the determinations of zinc by both proposed methods in plants supplied by M. PINTA (Comité Inter-Instituts pour l'Analyse Foliaire) show good accuracy and precision.Peer reviewe

Enhancement of lipase selectivity by site directed mutagenesis

Lipases belong to the α/β-hydrolase fold family and naturally catalyze the hydrolysis of fats and oils into glycerol and fatty acids. This class of enzymes displays numerous features that make them useful biocatalysts, including (i) broad substrate spectrum, (ii) excellent chemo-, regio- and stereoselectivity, (iii) high stability towards harsh reaction conditions, (iv) independence of cofactors and, furthermore, (v) a wide variety of lipases is commercially available. Due to all these advantages lipases have been widely applied in industrial processes such as dairy, baking, and detergent industry. Furthermore, they can be used for the production of trans-fatty acid free margarines and biodiesel [1-3]. However, despite their great applicability, each industrial application needs particular reaction conditions (e.g. substrate selectivity or stability towards temperature, pH and/or organic solvents) that should be borne by the biocatalyst. Therefore, protein engineering can be applied in order to obtain enzymes that meet the required parameters [4]. The present study focuses on the enhancement of lipase selectivity by protein engineering and its application for the enrichment of long chain fatty acids from natural oils, which are interesting building blocks for the chemical industry. Hence, a broad spectrum of commercial lipases was screened to identify those that already displayed the desired selectivity. Furthermore, lipases with interesting structural features were selected from literature as candidates for rational design [5, 6]. The most promising candidates were overexpressed in Pichia pastoris and Escherichia coli and subsequently purified to test their hydrolytic activity towards different p-nitrophenyl fatty acid esters. The best candidate found was subjected to molecular modelling to examine the potential hotspots to perform saturation mutagenesis. Three different amino acids present in the binding pocket were identified, allowing the design and creation of three combinatorial mutant libraries. Once the libraries were transformed into E. coli, the hydrolytic activity of more than 4500 clones was screened by using the fully automatized robotic platform LARA [7]. The most selective variants were chosen and used for confirmation of their activity and selectivity towards both, different chain length p-nitrophenyl fatty acid esters and several oil fractions. Acknowledgements: The COSMOS project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 635405. [1] Bornscheuer, U. T., Eur. J. Lipid Sci. Tech., 2014, 116, 1322-1331. [2] Borrelli, G. M. et al., Int. J. Mol. Sci., 2015, 16, 20774-20840. [3] Liu, H. et al., Biotechnol. Adv., 2014, 32, 382-389. [4] Zorn, K. et al., Prog. Lipid. Res., 2016, 63, 153-164. [5] Barriuso, J. et al., Biotechnol. Adv., 2016, 34, 874-885. [6] Brundiek, H. et al., Eur J Lipid Sci Technol, 2012, 114, 1148-1153. [7] Dörr, M. et al., Biotechnol. Bioeng., 2016, 113, 1421-1432

Atlas Toolkit: Fast registration of 3D morphological datasets in the absence of landmarks

Image registration is a gateway technology for Developmental Systems Biology, enabling computational analysis of related datasets within a shared coordinate system. Many registration tools rely on landmarks to ensure that datasets are correctly aligned; yet suitable landmarks are not present in many datasets. Atlas Toolkit is a Fiji/ImageJ plugin collection offering elastic group-wise registration of 3D morphological datasets, guided by segmentation of the interesting morphology. We demonstrate the method by combinatorial mapping of cell signalling events in the developing eyes of chick embryos, and use the integrated datasets to predictively enumerate Gene Regulatory Network states

Functional correlates of response inhibition in impulse control disorders in Parkinson’s disease

Impulse control disorder is a prevalent side-effect of Parkinson’s disease (PD) medication, with a strong negative impact on the quality of life of those affected. Although impulsivity has classically been associated with response inhibition deficits, previous evidence from PD patients with impulse control disorder (ICD) has not revealed behavioral dysfunction in response inhibition. In this study, 18 PD patients with ICD, 17 PD patients without this complication, and 15 healthy controls performed a version of the conditional Stop Signal Task during functional magnetic resonance imaging. Whole-brain contrasts, regions of interest, and functional connectivity analyses were conducted. Our aim was to investigate the neural underpinnings of two aspects of response inhibition: proactive inhibition, inhibition that has been prepared beforehand, and restrained inhibition, inhibition of an invalid inhibitory tendency. We observed that, in respect to the other two groups, PD patients with ICD exhibited hyperactivation of the stopping network bilaterally while performing proactive inhibition. When engaged in restrained inhibition, they showed hyperactivation of the left inferior frontal gyrus, an area linked to action monitoring. Restrained inhibition also resulted in changes to the functional co-activation between inhibitory regions and left inferior parietal cortex and right supramarginal gyrus. Our findings indicate that PD patients with ICD completed the inhibition task correctly, showing altered engagement of inhibitory and attentional areas. During proactive inhibition they showed bilateral hyperactivation of two inhibitory regions, while during restrained inhibition they showed additional involvement of attentional areas responsible for alerting and orientin

Mapping interactions with the chaperone network reveals factors that protect against tau aggregation.

A network of molecular chaperones is known to bind proteins ('clients') and balance their folding, function and turnover. However, it is often unclear which chaperones are critical for selective recognition of individual clients. It is also not clear why these key chaperones might fail in protein-aggregation diseases. Here, we utilized human microtubule-associated protein tau (MAPT or tau) as a model client to survey interactions between ~30 purified chaperones and ~20 disease-associated tau variants (~600 combinations). From this large-scale analysis, we identified human DnaJA2 as an unexpected, but potent, inhibitor of tau aggregation. DnaJA2 levels were correlated with tau pathology in human brains, supporting the idea that it is an important regulator of tau homeostasis. Of note, we found that some disease-associated tau variants were relatively immune to interactions with chaperones, suggesting a model in which avoiding physical recognition by chaperone networks may contribute to disease

Development of photocatalytic coatings for building materials with Bi2O3-ZnO nanoparticles

The aim of this study was to develop versatile coatings that can protect the stone surfaces of Ar-chitectural Heritage. Two different 3D media, namely superhydrophobic (SPHB) and hydrooleophobic (OHB), were utilized as host matrices for nanostructured photocatalysts (Bi2O3-ZnO 8/92). These photocatalysts were sensitive to visible light to enhance their efficiency when exposed to sunlight. To prevent the nanophotocatalyst from clumping together in the 3D media, non-ionic dispersant additives (Tween20, TritonX-100, and Brij35) were incorporated. The optimized suspensions were then applied to various substrates such as sandstone, limestone, and granite. The effectiveness of the coatings was assessed by evaluating the hydrophobicity, oleo-phobicity, and photocatalytic activity of the coated substrates. The Bi2O3-ZnO photocatalyst exhib-ited higher activity in the SPHB medium compared to the OHB medium. To simulate real-life con-ditions, the coated substrates were subjected to accelerated weathering tests to predict their dura-bility. Despite a significant reduction in their thickness, the coatings demonstrated sustained hy-drophobic efficiency and self-cleaning capability after the accelerated ageing tests

A Digital Framework to Build, Visualize and Analyze a Gene Expression Atlas with Cellular Resolution in Zebrafish Early Embryogenesis

A gene expression atlas is an essential resource to quantify and understand the multiscale processes of embryogenesis in time and space. The automated reconstruction of a prototypic 4D atlas for vertebrate early embryos, using multicolor fluorescence in situ hybridization with nuclear counterstain, requires dedicated computational strategies. To this goal, we designed an original methodological framework implemented in a software tool called Match-IT. With only minimal human supervision, our system is able to gather gene expression patterns observed in different analyzed embryos with phenotypic variability and map them onto a series of common 3D templates over time, creating a 4D atlas. This framework was used to construct an atlas composed of 6 gene expression templates from a cohort of zebrafish early embryos spanning 6 developmental stages from 4 to 6.3 hpf (hours post fertilization). They included 53 specimens, 181,415 detected cell nuclei and the segmentation of 98 gene expression patterns observed in 3D for 9 different genes. In addition, an interactive visualization software, Atlas-IT, was developed to inspect, supervise and analyze the atlas. Match-IT and Atlas-IT, including user manuals, representative datasets and video tutorials, are publicly and freely available online. We also propose computational methods and tools for the quantitative assessment of the gene expression templates at the cellular scale, with the identification, visualization and analysis of coexpression patterns, synexpression groups and their dynamics through developmental stages

Subregional 6-[18F]fluoro-ʟ-m-tyrosine Uptake in the Striatum in Parkinson's Disease

<p>Abstract</p> <p>Background</p> <p>In idiopathic Parkinson's disease (PD) the clinical features are heterogeneous and include different predominant symptoms. The aim of the present study was to determine the relationship between subregional aromatic l-amino acid decarboxylase (AADC) activity in the striatum and the cardinal motor symptoms of PD using high-resolution positron emission tomography (PET) with an AADC tracer, 6-[¹⁸F]fluoro-ʟ-<it>m</it>-tyrosine (FMT).</p> <p>Methods</p> <p>We assessed 101 patients with PD and 19 healthy volunteers. PD was diagnosed based on the UK Brain Bank criteria by two experts on movement disorders. Motor symptoms were measured with the Unified Parkinson's Disease Rating Scale (UPDRS). FMT uptake in the subregions of the striatum was analyzed using semi-automated software for region-of-interest demarcation on co-registered magnetic resonance images.</p> <p>Results</p> <p>In all PD patients, FMT uptake was decreased in the posterior putamen regardless of predominant motor symptoms and disease duration. Smaller uptake values were found in the putamen contralateral to the side with more affected limbs. The severity of bradykinesia, rigidity, and axial symptoms was correlated with the decrease of FMT uptake in the putamen, particularly in the anterior part. No significant correlation was observed between tremors and FMT uptake.</p> <p>Conclusions</p> <p>Decrease of FMT uptake in the posterior putamen appears to be most sensitive in mild PD and uptake in the anterior putamen may reflect the severity of main motor symptoms, except for tremor.</p

Real-time incidence of travel-related symptoms through a smartphone-based app remote monitoring system: a pilot study

Trip Doctor(R), a Smartphone-based app monitoring system, was developed to detect infections among travelers in real-time. For testing, 106 participants were recruited (62.2% male, mean age 36 years (SD = 11)). Majority of trips were for tourism and main destinations were in South East Asia. Mean travel duration was 14 days (SD = 10). Diarrhea was the most frequently reported symptom (15.5%). The system demonstrated adequate usability and is ready to be used on a larger scale

The Combination of Homocysteine and C-Reactive Protein Predicts the Outcomes of Chinese Patients with Parkinson's Disease and Vascular Parkinsonism

BACKGROUND: The elevation of plasma homocysteine (Hcy) and C-reactive protein (CRP) has been correlated to an increased risk of Parkinson's disease (PD) or vascular diseases. The association and clinical relevance of a combined assessment of Hcy and CRP levels in patients with PD and vascular parkinsonism (VP) are unknown. METHODOLOGY/PRINCIPAL FINDINGS: We performed a cross-sectional study of 88 Chinese patients with PD and VP using a clinical interview and the measurement of plasma Hcy and CRP to determine if Hcy and CRP levels in patients may predict the outcomes of the motor status, non-motor symptoms (NMS), disease severity, and cognitive declines. Each patient's NMS, cognitive deficit, disease severity, and motor status were assessed by the Nonmotor Symptoms Scale (NMSS), Mini-Mental State Examination (MMSE), the modified Hoehn and Yahr staging scale (H&Y), and the unified Parkinson's disease rating scale part III (UPDRS III), respectively. We found that 100% of patients with PD and VP presented with NMS. The UPDRS III significantly correlated with CRP (P = 0.011) and NMSS (P = 0.042) in PD patients. The H&Y was also correlated with Hcy (P = 0.002), CRP (P = 0.000), and NMSS (P = 0.023) in PD patients. In VP patients, the UPDRS III and H&Y were not significantly associated with NMSS, Hcy, CRP, or MMSE. Strong correlations were observed between Hcy and NMSS as well as between CRP and NMSS in PD and VP. CONCLUSIONS/SIGNIFICANCE: Our findings support the hypothesis that Hcy and CRP play important roles in the pathogenesis of PD. The combination of Hcy and CRP may be used to assess the progression of PD and VP. Whether or not anti-inflammatory medication could be used in the management of PD and VP will produce an interesting topic for further research