As the number falls,alternatives to the Hagberg–Perten falling number method: A review

This review examines the application, limitations, and potential alternatives to the Hagberg–Perten falling number (FN) method used in the global wheat industry for detecting the risk of poor end-product quality mainly due to starch degradation by the enzyme α-amylase. By viscometry, the FN test indirectly detects the presence of α-amylase, the primary enzyme that digests starch. Elevated α-amylase results in low FN and damages wheat product quality resulting in cakes that fall, and sticky bread and noodles. Low FN can occur from preharvest sprouting (PHS) and late maturity α-amylase (LMA). Moist or rainy conditions before harvest cause PHS on the mother plant. Continuously cool or fluctuating temperatures during the grain filling stage cause LMA. Due to the expression of additional hydrolytic enzymes, PHS has a stronger negative impact than LMA. Wheat grain with low FN/high α-amylase results in serious losses for farmers, traders, millers, and bakers worldwide. Although blending of low FN grain with sound wheat may be used as a means of moving affected grain through the marketplace, care must be taken to avoid grain lots from falling below contract-specified FN. A large amount of sound wheat can be ruined if mixed with a small amount of sprouted wheat. The FN method is widely employed to detect α-amylase after harvest. However, it has several limitations, including sampling variability, high cost, labor intensiveness, the destructive nature of the test, and an inability to differentiate between LMA and PHS. Faster, cheaper, and more accurate alternatives could improve breeding for resistance to PHS and LMA and could preserve the value of wheat grain by avoiding inadvertent mixing of high- and low-FN grain by enabling testing at more stages of the value stream including at harvest, delivery, transport, storage, and milling. Alternatives to the FN method explored here include the Rapid Visco Analyzer, enzyme assays, immunoassays, near-infrared spectroscopy, and hyperspectral imaging.     

Dreissenid veliger dynamics along a nearshore to offshore transect in Lake Michigan

Dreissenid mussel veligers compose a substantial component of pelagic biomass in the Great Lakes, yet their dynamics are poorly understood. To evaluate seasonal, spatial, and inter-annual variation in veliger density, we used a 64-μm mesh plankton net (2008, 2013–2016) and a 153-μm mesh plankton net (2007–2016) to collect dreissenid veligers at nearshore (15–25?m depth), transitional (45?m) and offshore (93–110?m) sites in southeast Lake Michigan during March–December. We also evaluated trends in density of recently settled mussels relative to veliger abundance and the density of the standing stock of adult mussels. Veliger density peaked during both summer and fall at all sites, but peak densities in summer were generally higher nearshore, whereas peak densities in the fall were generally higher offshore. The density of veligers in the 153-μm net was overall 28% of that in the 64-μm net, but there was high variability in this comparison among months. Smaller veligers were much more abundant in the 64-μm net, but there was little difference in the size distribution and abundance between nets for the 210–300?μm size classes. Thus, the 153-μm net could still be a useful tool for assessing density trends of larger veligers just prior to their settlement. Newly settled mussels (≤2?mm) were most abundant in summer or fall at the nearshore and offshore sites but were nearly absent at the transitional site despite the high density of veligers there. Factors other than veliger density must play an important role in mussel recruitment.     

A boronic acid-based fluorescent hydrogel for monosaccharide detection

A boronic acid-based anthracene fluorescent probe was functionalised with an acrylamide unit to incorporate into a hydrogel system for monosaccharide detection. In solution, the fluorescent probe displayed a strong fluorescence turn-on response upon exposure to fructose, and an expected trend in apparent binding constants, as judged by a fluorescence response where D-fructose>D-galactose>D-mannose>D-glucose. The hydrogel incorporating the boronic acid monomer demonstrated the ability to detect monosaccharides by fluorescence with the same overall trend as the monomer in solution with the addition of D-fructose resulting in a 10-fold enhancement (≤0.25 mol/L).     

An Atomistic Understanding of Allosteric Inhibition of Glutamate Racemase: a Dampening of Native Activation Dynamics

Glutamate racemases (GR) are members of the family of bacterial enzymes known as cofactor-independent racemases and epimerases and catalyze the stereoinversion of glutamate. D-amino acids are universally important for the proper construction of viable bacterial cell walls, and thus have been repeatedly validated as attractive targets for novel antimicrobial drug design. Significant aspects of the mechanism of this challenging stereoinversion remain unknown. The current study employs a combination of MD and QM/MM computational approaches to show that the GR from H. pylori must proceed via a pre-activation step, which is dependent on the enzyme's flexibility. This mechanism is starkly different from previously proposed mechanisms. These findings have immediate pharmaceutical relevance, as the H. pylori GR enzyme is a very attractive allosteric drug target. The results presented in this study offer a distinctly novel understanding of how AstraZeneca's lead series of inhibitors cripple the H. pylori GR's native motions, via prevention of this critical chemical pre-activation step. Our experimental studies, using SPR, fluorescence and NMR WaterLOGSY, show that H. pylori GR is not inhibited by the uncompetitive mechanism originally put forward by Lundqvist et al.. The current study supports a deep connection between native enzyme motions and chemical reactivity, which has strong relevance to the field of allosteric drug discovery.     

Damage evolution in SiC/SiC unidirectional composites by X-ray tomography

Melt infiltrated SiC/SiC ceramic matrix composite unidirectional (UD) composite specimens were imaged under load using X-ray microtomography techniques in order to visualize the evolution of damage accumulation and to quantify damage mechanisms within the composite such as matrix cracking and fiber breaking. The data obtained from these in situ tensile tests were used in comparison with current models and literature results. Three-dimensional (3D) tomography images were used to measure the location and spacing of matrix cracking that occurred at increasing stress increments during testing within two UD composite specimens. The number of broken fibers and the location of each fiber break gap that occurred within the volume of both specimens were also quantified. The 3D locations of fiber breaks were correlated with the location of each matrix crack within the volume of the specimen and it was found that at the stress scanned directly before failure, most of the fiber breaks occur within 100 microns of a matrix crack.     

ADVANCES IN HIGH-RESOLUTION MALDI MASS SPECTROMETRY FOR NEUROBIOLOGY

Research in the field of neurobiology and neurochemistry has seen a rapid expansion in the last several years due to advances in technologies and instrumentation, facilitating the detection of biomolecules critical to the complex signaling of neurons. Part of this growth has been due to the development and implementation of high-resolution Fourier transform (FT) mass spectrometry (MS), as is offered by FT ion cyclotron resonance (FTICR) and Orbitrap mass analyzers, which improves the accuracy of measurements and helps resolve the complex biological mixtures often analyzed in the nervous system. The coupling of matrix-assisted laser desorption/ionization (MALDI) with high-resolution MS has drastically expanded the information that can be obtained with these complex samples. This review discusses notable technical developments in MALDI-FTICR and MALDI-Orbitrap platforms and their applications toward molecules in the nervous system, including sequence elucidation and profiling with de novo sequencing, analysis of post-translational modifications, in situ analysis, key advances in sample preparation and handling, quantitation, and imaging. Notable novel applications are also discussed to highlight key developments critical to advancing our understanding of neurobiology and providing insight into the exciting future of this field. © 2020 John Wiley & Sons Ltd. Mass Spec Rev     

Discovery of Quinoline‐Derived Trifluoromethyl Alcohols,Determination of Their in vivo Toxicity and Anticancer Activity in a Zebrafish Embryo Model

In this study the rational design, synthesis, and anticancer activity of quinoline‐derived trifluoromethyl alcohols were evaluated. Members of this novel class of trifluoromethyl alcohols were identified as potent growth inhibitors in a zebrafish embryo model. Synthesis of these compounds was carried out with an sp³‐C?H functionalization strategy of methyl quinolines with trifluoromethyl ketones. A zebrafish embryo model was also used to explore the toxicity of ethyl 4,4,4‐trifluoro‐3‐hydroxy‐3‐(quinolin‐2‐ylmethyl)butanoate ( 1 ), 2‐benzyl‐1,1,1‐trifluoro‐3‐(quinolin‐2‐yl)propan‐2‐ol ( 2 ), and trifluoro‐3‐(isoquinolin‐1‐yl)‐2‐(thiophen‐2‐yl)propan‐2‐ol ( 3 ). Compounds 2 and 3 were found to be more toxic than compound 1 ; apoptotic staining assays indicated that compound 3 causes increased cell death. In vitro cell proliferation assays showed that compound 2 , with an LC₅₀ value of 14.14 μm , has more potent anticancer activity than cisplatin. This novel class of inhibitors provides a new direction in the discovery of effective anticancer agents.     

Identification of Sortilin Alternatively Spliced Variants in Mouse 3T3L1 Adipocytes

Type 2 diabetes mellitus is a metabolic disorder defined by systemic insulin resistance. Insulin resistance in adipocytes, an important regulator of glucose metabolism, results in impaired glucose uptake. The trafficking protein, sortilin, regulates major glucose transporter 4 (Glut4) movement, thereby promoting glucose uptake in adipocytes. Here, we demonstrate the presence of an alternatively spliced sortilin variant (Sort^17b), whose levels increase with insulin resistance in mouse 3T3L1 adipocytes. Using a splicing minigene, we show that inclusion of alternative exon 17b results in the expression of Sort^17b splice variant. Bioinformatic analysis indicated a novel intrinsic disorder region (IDR) encoded by exon 17b of Sort^17b. Root mean square deviation (RMSD) and root mean square fluctuation (RMSF) measurements using molecular dynamics demonstrated increased flexibility of the protein backbone within the IDR. Using protein–protein docking and co-immunoprecipitation assays, we show robust binding of Glut4 to Sort^17b. Further, results demonstrate that over-expression of Sort^17b correlates with reduced Glut4 translocation and decreased glucose uptake in adipocytes. The study demonstrates that insulin resistance in 3T3L1 adipocytes promotes expression of a novel sortilin splice variant with thus far unknown implications in glucose metabolism. This knowledge may be used to develop therapeutics targeting sortilin variants in the management of type 2 diabetes and metabolic syndrome.