Multicomponent Bioluminescence Imaging with Naphthylamino Luciferins

Bioluminescent tools have been used for decades to image processes in complex tissues and preclinical models. However, few distinct probes are available to probe multicellular interactions. We and others are addressing this limitation by engineering new luciferases that can selectively process synthetic luciferin analogues. In this work, we explored naphthylamino luciferins as orthogonal bioluminescent probes. Three analogues were prepared using an optimized synthetic route. The luciferins were found to be robust emitters with native luciferase in vitro and in cellulo. We further screened the analogues against libraries of luciferase mutants to identify unique enzyme-substrate pairs. The new probes can be used in conjunction with existing bioluminescent tools for multi-component imaging.     

Lipid products formed during desaturation of [1-14C] stearyl CoA by hen liver microsomes

A number of lipid products are formed during the desaturation of stearyl CoA by hen liver microsomes. This article presents an analysis of the products formed when [1-¹⁴C] stearyl CoA is incubated with hen liver microsomes for various time periods. [1-¹⁴C] Oleyl CoA was the first radioactive unsaturated product formed. Synthesis of phospholipids containing [1-¹⁴C] oleic acid occurs only after the desaturase activity has begun to decline. The specific radioactivity of [1-¹⁴C] oleyl CoA was similar to the specific radioactivity of [1-¹⁴C] stearyl CoA at all time periods tested. The specific radioactivities of [1-¹⁴C] oleic acid and phospholipids containing [1-¹⁴C] oleic acid were much lower than that of the [1-¹⁴C] stearyl CoA.     

Differently sized native milk fat globules separated by microfiltration: fatty acid composition of the milk fat globule membrane and triglyceride core

The aim of this study was to characterize the fatty acid composition of the core and membrane of differently sized milk fat globules separated by microfiltration, which can now be used to manufacture dairy products. Native milk fat globules of various mean diameters, ranging from d₄₃ = 2.3 µm to 8.0 µm, were obtained using microfiltration of raw whole milk. After milk fat globule washing, the milk fat globule membrane (MFGM) and the triglyceride core (TC) were separated by manual churning. After total lipid extraction from each fraction, their fatty acid composition was characterized using methyl ester analysis by gas chromatography. Regardless of season, no significant differences were observed in the fatty acid composition of the MFGM phospholipids. Conversely, significant differences were found in the fatty acid composition of TC; particularly, small fat globule TC contained more medium‐chain fatty acids and less stearic acid than large fat globule TC. These results show that the previously observed differences in total fatty acid composition among differently sized milk fat globules are due to their triglyceride composition and MFGM amount rather than to the composition of the MFGM.     

Regulation of phosphatidylinositol turnover,calcium metabolism and enzyme secretion by phorbol dibutyrate in neutrophils

The action of the tumor promoter, phorbol 12,13-dibutyrate (PDBu), on rabbit peritoneal and human neutrophils is associated with stimulation of¹⁴C-arachidonic acid incorporation into phospholipids within 1–2 min. Stimulated¹⁴C-arachidonate incorporation was relatively selective for phosphatidylinositol (PI) in rabbit neutrophils. In contrast, the secretory response of human neutrophils to PDBu coincided with stimulated label incorporation into phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA) and PI. Significant increases in label incorporation were observed with PDBu concentrations as low as 2 nM, and the dose response of stimulated label incorporation paralleled that of evoked lysozyme secretion. A parallel, but partial, inhibition of PDBu-stimulated PI labeling and enzyme release was observed after exposing rabbit neutrophils to calcium-deprived medium, whereas calcium deprivation failed to significantly depress either of these stimulant actions of PDBu in human neutrophils. Further, in rabbit neutrophils PDBu elicited an increase in cell associated⁴⁵Ca. However, PDBu was unable to promote the incorporation of³²P orthophosphate into PI or enhance phospholipase A₂ activity in broken cells. These findings suggest that one expression of the interaction between phorbol esters and their receptors on neutrophils involves the turnover of arachidonic acid in phospholipids. This stimulated turnover of arachidonate may be a critical step in the cascade of events associated with neutrophil activation.     

New associative systems based on alkylated hyaluronic acid. Synthesis and aqueous solution properties

This paper concerns the preparation of new water-soluble alkylated derivatives of hyaluronan (HA); these derivatives were synthesized under mild aqueous and well controlled conditions. The first step was to prepare a HA derivative selectively modified with adipic dihydrazide (HA-ADH) with a substitution degree of 0.08; then, alkyl chains with different chain lengths were introduced with a substitution degree around 0.05. These polymers being still water-soluble exhibit associating properties in the presence of external salt. Formation of hydrophobic domains is demonstrated from fluorescence measurements in the presence of pyrene as a probe. Then, it is shown that the viscosity of polymer solutions in 0.1 M NaCl becomes non-Newtonian for HA grafted with C-10 and C-12 chains and increases rapidly with the polymer concentration over 1 g/L. When the polymer concentration increases, a gel-like behaviour was observed from rheological measurements with the G′ modulus larger than G″ in all the frequency range covered. The dependence of the shear stress on shear rate applied according to a continuous linear ramp provided evidence of the deformation or rupture of aggregates. The latter were shown to reform slowly. At the end, the influence of temperature was examined: first a slight increase in the modulus was observed followed by a decrease corresponding to reinforcement of the hydrophobic interactions.     

Boronic acid-functionalized nanoparticles: synthesis by microemulsion polymerization and application as a re-usable optical nanosensor for carbohydrates

Translucent boronic acid-carrying nanolatexes are prepared by copolymerisation of vinylbenzyl chloride in microemulsion followed by post functionalisation with 3-aminophenylboronic acid. The diol binding capacity and selectivity of the phenylboronic acid remain unchanged after anchoring to polymer nanoparticles (16 nm diameter, 0.22 mmol/g). The binding of a catechol dye, Alizarin Red S (ARS), to the boronic acid-carrying nanoparticles affords a colored, stable, nanolatex which is used for the selective visual detection of fructose in a competitive assay. The binding of fructose at pH 8.2 is readily evidenced by a color change and monitored by UV/Vis spectroscopy. The nanosensor can be regenerated in acidic medium. The entrapment of the nanolatex in a dialysis cell provides a re-usable support for binding and optical detection.     

pH sensitive phosphate crosslinked films of starch-carboxymethyl cellulose

This work aims to develop hydrogel films of starch and carboxymethyl cellulose (CMC) crosslinked with sodium trimetaphosphate (STMP) and to characterize some of their properties. Starch and STMP (S/T), starch and CMC (S/C), and mixed (S/T/C) films were prepared by casting. The degree of substitution, morphology, swelling degree, FTIR, mechanical properties, and sorption isotherms were studied. Reticulated samples (S/T and S/T/C) showed the same degree of substitution (0.050 ± 0.001). All films presented homogeneous morphology, but the mixed film showed greater roughness. Crosslinking increased the swelling capacity of the mixed hydrogel at pH 7, although it remained decreased concerning the S/T hydrogel. However, this property was sensitive to pH variations. The mixed film (S/T/C) showed greater mechanical resistance. The casting process was efficient to produce hydrogel films of starch/CMC crosslinked with STMP and the general results demonstrated the advantages of the mixed hydrogel.     

Influence of nucleation and growth mechanisms on the heat deflection temperature of a reactively processed polypropylene nanocomposite

The development of a reactively processed polypropylene nanocomposite (PPNC) with consequential improvements in the heat deflection temperature (HDT), Vicat softening temperature (VST), and crystallization peak temperature (T_c) is reported herein. Neat PP without nanoclay was also reactively processed to elucidate the effects of fillers on the improvement in physical properties. The results show a considerable improvement in the HDT of PPNC (77.9 °C) compared to those of neat PP (62.6 °C) and reactively processed branched PP (BPP; 69.2 °C). Moreover, the T_c of PP in PPNC improved by ~14% compared to that of neat PP. Various models of nonisothermal crystallization kinetics were employed to elucidate the nucleation and crystal growth mechanisms, and to correlate them with the observed HDT improvement in PPNC. Thermal transitions investigated by modulated differential scanning calorimetry explained the changes observed in the VSTs of all the samples. To the best of our knowledge, this is the first report on a significant improvement in HDT along with a marked increase in Tc. Such simultaneous improvements in HDT, VST, and T_c are highly desirable for applications involving the use of PP-based materials in rigid packaging.     

Adaptive infill sampling criterion for multi-fidelity optimization based on Gappy-POD

This paper presents a reformulation of the “Gappy Proper Orthogonal Decomposition” (Gappy-POD) multi-fidelity modeling approach and proposes an enrichment criterion associated with an adaptive infill algorithm. The latter is here applied to the study of the flight domain of the RAE-2822 transonic airfoil at two different levels of accuracy to demonstrate its ability to detect areas in a two-dimensional design space where the surrogate model needs improvement to better drive the optimization process.     

Making a Clean Sweep of Cultural Heritage

Digitization brings about new ways of analyzing data from cultural heritage areas. Automatic error detection, as input to semiautomatic error correction, is one type of analysis that can be found high on the priority list of cultural heritage data managers and researchers. We describe a general approach to cleaning cultural heritage databases. We present four case studies on databases from different cultural heritage institutions, and describe an information system in which we embed our error detector in a larger framework, enabling researchers to access, check, and correct their data more easily than before.