The health benefits of human milk oligosaccharides (HMOs) make them attractive targets as supplements for infant formula milks. However, HMO synthesis is still challenging and only two HMOs have been marketed. Engineering glycoside hydrolases into transglycosylases may provide biocatalytic routes to the synthesis of complex oligosaccharides. Lacto-N-biosidase from Bifidobacterium bifidum (LnbB) is a GH20 enzyme present in the gut microbiota of breast-fed infants that hydrolyzes lacto-N-tetraose (LNT), the core structure of the most abundant type I HMOs. Here we report a mutational study in the donor subsites of the substrate binding cleft with the aim of reducing hydrolytic activity and conferring transglycosylation activity for the synthesis of LNT from p-nitrophenyl β-lacto-N-bioside and lactose. As compared with the wt enzyme with negligible transglycosylation activity, mutants with residual hydrolase activity within 0.05% to 1.6% of the wild-type enzyme result in transglycosylating enzymes with LNT yields in the range of 10–30%. Mutations of Trp394, located in subsite -1 next to the catalytic residues, have a large impact on the transglycosylation/hydrolysis ratio, with W394F being the best mutant as a biocatalyst producing LNT at 32% yield. It is the first reported transglycosylating LnbB enzyme variant, amenable to further engineering for practical enzymatic synthesis of LNT. 相似文献
We report a wavelength-switchable fiber ring laser that includes a Bragg grating-based acoustooptic superlattice modulator. Wavelength switching of the laser emission is achieved by controlling the peak reflectivity of the different reflection bands that appear on both sides of the Bragg wavelength when a longitudinal acoustic wave propagates along the grating. 相似文献
We describe a novel microfluidic perfusion system for high-resolution microscopes. Its modular design allows pre-coating of
the coverslip surface with reagents, biomolecules, or cells. A poly(dimethylsiloxane) (PDMS) layer is cast in a special molding
station, using masters made by photolithography and dry etching of silicon or by photoresist patterning on glass or silicon.
This channel system can be reused while the coverslip is exchanged between experiments. As normal fluidic connectors are used,
the link to external, computer-programmable syringe pumps is standardized and various fluidic channel networks can be used
in the same setup. The system can house hydrogel microvalves and microelectrodes close to the imaging area to control the
influx of reaction partners. We present a range of applications, including single-molecule analysis by fluorescence correlation
spectroscopy (FCS), manipulation of single molecules for nanostructuring by hydrodynamic flow fields or the action of motor
proteins, generation of concentration gradients, trapping and stretching of live cells using optical fibers precisely mounted
in the PDMS layer, and the integration of microelectrodes for actuation and sensing. 相似文献
The present paper tries to contribute to quantifying the relationship between the amount of corrosion and cover cracking. The variables studied were: cover/diameter (c/ø), proportions of cement, w/c, cast position of the bar, transverse reinforcement and corrosion rate. The corrosion is accelerated by applying constant currents causing the rebar to act as an anode. The results indicate that the cracking process develops in two steps: generation and propagation. Radius losses of about 15–50 μm are necessary to generate the first visible crack (<0.1 mm width). The propagation follows a behaviour of the type: w (crack width in mm)=a+bx (radius loss in μm). 相似文献
In this paper, analytical functions for the estimation of the temperature-dependent behaviors of poorly and highly dispersed graphene oxide reinforced nanocomposite (GORNC) materials are studied in the framework of a machine learning-based approach. The validity of the presented models is shown comparing the results achieved from this modeling with those reported in the open literature. Also, the application of the obtained functions in solving the thermal buckling problem of beams constructed from such nanocomposites is demonstrated based on an energy-based method incorporated with a shear deformable beam hypothesis. The verification of the results indicates that the presented mechanical model can approximate the buckling behaviors of nanocomposite beams with remarkable precision. It can be realized from the results that the temperature plays an indispensable role in the determination of the buckling load which can be endured by the nanocomposite structure.
Methamphetamine is, worldwide, one of the most consumed drugs of abuse. One important side effect is neurodegeneration leading to a decrease in life expectancy. The aim of this paper was to check whether the drug affects one of the receptors involved in neurodegeneration/neuroprotection events, namely the adenosine A2A receptor (A2AR). First, we noticed that methamphetamine does not affect A2A functionality if the receptor is expressed in a heterologous system. However, A2AR becomes sensitive to the drug upon complexes formation with the cannabinoid CB1 receptor (CB1R) and the sigma 1 receptor (σ1R). Signaling via both adenosine A2AR and cannabinoid CB1R was affected by methamphetamine in cells co-expressing the two receptors. In striatal primary cultures, the A2AR–CB1R heteromer complex was detected and methamphetamine not only altered its expression but completely blocked the A2AR- and the CB1R-mediated activation of the mitogen activated protein kinase (MAPK) pathway. In conclusion, methamphetamine, with the participation of σ1R, alters the expression and function of two interacting receptors, A2AR, which is a therapeutic target for neuroprotection, and CB1R, which is the most abundant G protein-coupled receptor (GPCR) in the brain. 相似文献
To develop a synthesis of tritiated cyclopropene fatty acids (CPFA), compounds that should prove useful for affinity labeling
of desaturases in insect pheromone biosynthetic studies, a series of novel, selectively deuterated CPFA analogues was prepared
and characterized. In methyl [16-2H]12,13-methylene-12-hexadecenoate, the incorporation of deuterium was achieved by treatment of the corresponding ω-chloro
derivative with sodium borodeuteride in dimethylsulfoxide at 70°C for 24 h (67% yield) following conventional procedures.
Alkylation of the tetrahydropyranyl derivative of 13-tridecynol in the presence of lithium diisopropylamide in tetrahydrofuran
at −20°C with 1-chloro-3-iodopropane in hexamethylphosphoramide, followed by Jones oxidation of the crude product, yielded
16-chloro-12-hexadecynoic acid (54%), which was esterified to the corresponding methyl ester by treatment with potassium carbonate
and methyl iodide in dimethylformamide. Treatment of this acetylenic ester with ethyldiazoacetate in the presence of activated
copper-bronze as catalyst followed by hydrolysis in KOH solution at room temperature yielded 16-chloro-12,13-(carboxymethylene)-12-hexadecenoic
acid. This diacid was treated with excess oxalyl chloride to give the corresponding diacyl chloride, which was decarbonylated
in a diethyl ether solution with zinc chloride, and the cyclopropenium ions thus formed were added at −40°C to a methanolic
sodium hydroxide solution of sodium borohydride to give methyl 16-chloro-12,13-methylene-12-hexadecenoate. Analogous procedures
were followed to prepare methyl [17-2H]10,11-methylene-10-hexadecenoate, methyl [17-2H]11,12-methylene-11-hexadecenoate and methyl [17-2H]12,13-methylene-12-hexadecenoate from the corresponding diacids using sodium borodeuteride in the reduction of the cyclopropenium
ions. Alternatively, methyl [2,2,3,3-2H4]hexadecynoate, prepared by reaction of methyl 2,11-hexadecadiynoate with magnesium in deuterated methanol at room temperature,
was submitted to the above cyclopropenylation and reductive decarbonylation sequence to give methyl [2,2,3,3,17-2H5]-11,12-methylene-11-hexadecenoate. In summary, complementary methods for the selective incorporation of one to five deuterium
atoms into cyclopropene fatty acids, at different sites, in moderate to high yields have been developed. The methods should
easily be applicable to the preparation of the corresponding tritiated analogues. 相似文献
A variant phenotype of nonketotic hyperglycinemia has been described by our group associated with pulmonary hypertension. The aim of this study is to investigate the cerebrospinal fluid proteomes to get an insight into this neurodegenerative process producing leukoencephalopathy with white matter spongiform degeneration. DIGE and MALDI-TOF-TOF analyses were performed to carry out the proteomic study of four patients against three normal controls and one additional control of a classical nonketotic hyperglycinemia. The differential proteomic analysis showed a displacement of some series of spots toward the acidic side. The shifted proteins showed a high degree of carbonylation and increased methionine sulfoxidation was found in cystatin C and in vitamin-D-binding protein. These findings in addition to the increase of serum malondialdehyde concentration provide evidence of an oxidative stress in the patients under study, which is probably systemic rather than mainly confined to the CNS. The similarities of our findings with those found in other neurodegenerative diseases suggest that oxidative damage is commonly involved in these pathologies. DIGE technology improves the 2-D PAGE differential analysis and it is suitable in proteomic studies with a small number of cases. 相似文献