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Carbohydrates comprise the largest fraction of most diets and exert a profound impact on health. Components such as simple sugars and starch supply energy, while indigestible components, deemed dietary fiber, reach the colon to provide food for the tens of trillions of microbes that make up the gut microbiota. The interactions between dietary carbohydrates, our gastrointestinal tracts, the gut microbiome and host health are dictated by their structures. However, current methods for analysis of food glycans lack the sensitivity, specificity and throughput needed to quantify and elucidate these myriad structures. This protocol describes a multi-glycomic approach to food carbohydrate analysis in which the analyte might be any food item or biological material such as fecal and cecal samples. The carbohydrates are extracted by ethanol precipitation, and the resulting samples are subjected to rapid-throughput liquid chromatography (LC)-tandem mass spectrometry (LC-MS/MS) methods. Quantitative analyses of monosaccharides, glycosidic linkages, polysaccharides and alcohol-soluble carbohydrates are performed in 96-well plates at the milligram scale to reduce the biomass of sample required and enhance throughput. Detailed stepwise processes for sample preparation, LC-MS/MS and data analysis are provided. We illustrate the application of the protocol to a diverse set of foods as well as different apple cultivars and various fermented foods. Furthermore, we show the utility of these methods in elucidating glycan–microbe interactions in germ-free and colonized mice. These methods provide a framework for elucidating relationships between dietary fiber, the gut microbiome and human physiology. These structures will further guide nutritional and clinical feeding studies that enhance our understanding of the role of diet in nutrition and health. Key points It is important to understand how carbohydrates are digested—both by human enzymes and by microorganisms present in the gut. This protocol is designed to characterize and quantify food and fecal polysaccharides at the monosaccharide, linkage and polysaccharide level. Analysis is performed by LC-MS/MS. Higher-throughput sample preparation in 96-well plates is possible by using a custom-made clamp to hold the plate lids closed during heating. Complex carbohydrates that are not broken down by human enzymes are food sources for gut microbiota. Toward understanding this process, this protocol describes the quantitative analysis of carbohydrates in food and fecal samples by using LC-MS/MS.

A novel strategy for the qualitative analysis of carbohydrates is developed, utilizing a direct catalytic fuel cell (DCFC) as a sensor, combined with chemometric tools for processing the resulting response curves. Specifically, carbohydrate solutions were incubated with yeast to produce alcohol, and the corresponding current decay trends were measured using a direct catalytic fuel cell designed for ethanol detection. Multiple data processing approaches were then evaluated. Initially, the entire set of data points from the response curves was analyzed using principal component analysis (PCA). To reduce analysis time, chemometric processing was subsequently restricted to the initial portion of the response curves. Finally, to enhance the results, the current decay curves were analyzed in conjunction with the linear fitting parameters derived from the quasi-linear region of the initial response curves, utilizing the common dimension (ComDim) algorithm.

There has been much concern regarding the role of dietary fructose in the development of metabolic diseases. This concern arises from the continuous increase in fructose (and total added caloric sweeteners consumption) in recent decades, and from the increased use of high-fructose corn syrup (HFCS) as a sweetener. A large body of evidence shows that a high-fructose diet leads to the development of obesity, diabetes, and dyslipidemia in rodents. In humans, fructose has long been known to increase plasma triglyceride concentrations. In addition, when ingested in large amounts as part of a hypercaloric diet, it can cause hepatic insulin resistance, increased total and visceral fat mass, and accumulation of ectopic fat in the liver and skeletal muscle. These early effects may be instrumental in causing, in the long run, the development of the metabolic syndrome. There is however only limited evidence that fructose per se, when consumed in moderate amounts, has deleterious effects. Several effects of a high-fructose diet in humans can be observed with high-fat or high-glucose diets as well, suggesting that an excess caloric intake may be the main factor involved in the development of the metabolic syndrome. The major source of fructose in our diet is with sweetened beverages (and with other products in which caloric sweeteners have been added). The progressive replacement of sucrose by HFCS is however unlikely to be directly involved in the epidemy of metabolic disease, because HFCS appears to have basically the same metabolic effects as sucrose. Consumption of sweetened beverages is however clearly associated with excess calorie intake, and an increased risk of diabetes and cardiovascular diseases through an increase in body weight. This has led to the recommendation to limit the daily intake of sugar calories.

Terahertz (THz=1012Hz) radiation has attracted wide attention for its unprecedented sensing ability and its noninvasive and nonionizing properties. Tremendous strides in THz instrumentation have prompted impressive breakthroughs in THz biomedical research. Here, we review the current state of THz spectroscopy and imaging in various biomedical applications ranging from biomolecules, including DNA/RNA, amino acids/peptides, proteins, and carbohydrates, to cells and tissues. We also address the potential biological effects of THz radiation during its biological applications and propose future prospects for this cutting-edge technology. THz spectroscopy has proven to be an innovative tool for providing new insights into the hydration shell in the solvation dynamics of protein solutions. THz in-line digital holography, THz near-field imaging modality, and THz endoscope prototypes have been utilized to identify abnormal tissues faster and more accurately. Increasing applications of artificial modeling and numerical computation are becoming essential supplements for THz biological effect studies.

Carbohydrates provide the building blocks for plant structures as well as versatile resources for metabolic processes. The nonstructural carbohydrates (NSC), mainly sugars and starch, fulfil distinct functional roles, including transport, energy metabolism and osmoregulation, and provide substrates for the synthesis of defence compounds or exchange with symbionts involved in nutrient acquisition or defence. At the whole-plant level, NSC storage buffers the asynchrony of supply and demand on diel, seasonal or decadal temporal scales and across plant organs. Despite its central role in plant function and in stand-level carbon cycling, our understanding of storage dynamics, its controls and response to environmental stresses is very limited, even after a century of research. This reflects the fact that often storage is defined by what we can measure, that is, NSC concentrations, and the interpretation of these as a proxy for a single function, storage, rather than the outcome of a range of NSC source and sink functions. New isotopic tools allow direct quantification of timescales involved in NSC dynamics, and show that NSC-C fixed years to decades previously is used to support tree functions. Here we review recent advances, with emphasis on the context of the interactions between NSC, drought and tree mortality.

A strain of embryonic human kidney cells (HEK293) was transiently co-transfected with the expression vectors coding for the α- and β-subunits of human thyroid-stimulating hormone (hTSH), and, for the first time, a human cell-derived recombinant hTSH was synthesized and extensively characterized. The purification strategy involving two steps provided an overall yield of 55% and a purity level > 90%. The purified material (hTSH-HEK) was analyzed and compared to a CHO-derived recombinant preparation (hTSH-CHO) and to a pituitary-derived (hTSH-Pit) preparation. The three preparations showed an equivalent purity (> 95%) with a hTSH-HEK molecular mass 2.1% lower than that of hTSH-CHO and 2.7% higher than that of hTSH-Pit. Remarkable differences were found in the carbohydrate moiety, the lowest sialic acid content and highest fucose content being observed in hTSH-HEK. In vivo biological activity was confirmed for the three preparations, the hTSH-HEK bioactivity being 39 and 16% lower than those of hTSH-CHO and hTSH-Pit, respectively. The hTSH-HEK circulatory half-life (t1/2) was also shorter than those of hTSH-CHO (1.5-fold) and hTSH-Pit (1.2-fold). According to these findings, HEK-293-derived hTSH can be considered to be useful for clinical applications, in view as well of its human origin and particular carbohydrate composition.

Metabolic labeling of the cell surface with a caged azide sugar enabled cleavage-mediated activation by enzymes overexpressed in cancer cells, allowing enhanced targeted delivery of a doxorubicin conjugate through copper-free click chemistry. Distinguishing cancer cells from normal cells through surface receptors is vital for cancer diagnosis and targeted therapy. Metabolic glycoengineering of unnatural sugars provides a powerful tool to manually introduce chemical receptors onto the cell surface; however, cancer-selective labeling still remains a great challenge. Herein we report the design of sugars that can selectively label cancer cells both in vitro and in vivo . Specifically, we inhibit the cell-labeling activity of tetraacetyl- N -azidoacetylmannosamine (Ac 4 ManAz) by converting its anomeric acetyl group to a caged ether bond that can be selectively cleaved by cancer-overexpressed enzymes and thus enables the overexpression of azido groups on the surface of cancer cells. Histone deacetylase and cathepsin L-responsive acetylated azidomannosamine, one such enzymatically activatable Ac 4 ManAz analog developed, mediated cancer-selective labeling in vivo , which enhanced tumor accumulation of a dibenzocyclooctyne–doxorubicin conjugate via click chemistry and enabled targeted therapy against LS174T colon cancer, MDA-MB-231 triple-negative breast cancer and 4T1 metastatic breast cancer in mice.

The branched structure and stereoisomerism of carbohydrates make them difficult to analyse; here, ion mobility–mass spectrometry is used to distinguish unambiguously between synthetic trisaccharides that differ in connectivity or configuration. A new approach to carbohydrate analysis The branched structure and stereoisomerism of carbohydrates make them difficult to analyse, especially if they are synthetic. Peter Seeberger and colleagues demonstrate here that ion mobility–mass spectrometry (IM–MS), a method that separates molecules according to mass, charge, size, and shape, can unambiguously identify glycan regio- and stereoisomers. Six synthetic carbohydrate isomers that differ in composition, connectivity, or configuration were analysed. The analysis is rapid and requires only small amounts of sample, suggesting that IM–MS will be an exceptionally effective tool for the analysis of complex carbohydrates. Carbohydrates are ubiquitous biological polymers that are important in a broad range of biological processes 1 , 2 , 3 . However, owing to their branched structures and the presence of stereogenic centres at each glycosidic linkage between monomers, carbohydrates are harder to characterize than are peptides and oligonucleotides 4 . Methods such as nuclear magnetic resonance spectroscopy can be used to characterize glycosidic linkages, but this technique requires milligram amounts of material and cannot detect small amounts of coexisting isomers 5 . Mass spectrometry, on the other hand, can provide information on carbohydrate composition and connectivity for even small amounts of sample, but it cannot be used to distinguish between stereoisomers 6 . Here, we demonstrate that ion mobility–mass spectrometry—a method that separates molecules according to their mass, charge, size, and shape—can unambiguously identify carbohydrate linkage-isomers and stereoisomers. We analysed six synthetic carbohydrate isomers that differ in composition, connectivity, or configuration. Our data show that coexisting carbohydrate isomers can be identified, and relative concentrations of the minor isomer as low as 0.1 per cent can be detected. In addition, the analysis is rapid, and requires no derivatization and only small amounts of sample. These results indicate that ion mobility–mass spectrometry is an effective tool for the analysis of complex carbohydrates. This method could have an impact on the field of carbohydrate synthesis similar to that of the advent of high-performance liquid chromatography on the field of peptide assembly in the late 1970s.

Chickpea (Cicer arietinum L.) is an important pulse crop grown and consumed all over the world, especially in the Afro-Asian countries. It is a good source of carbohydrates and protein, and protein quality is considered to be better than other pulses. Chickpea has significant amounts of all the essential amino acids except sulphur-containing amino acids, which can be complemented by adding cereals to the daily diet. Starch is the major storage carbohydrate followed by dietary fibre, oligosaccharides and simple sugars such as glucose and sucrose. Although lipids are present in low amounts, chickpea is rich in nutritionally important unsaturated fatty acids such as linoleic and oleic acids. β-Sitosterol, campesterol and stigmasterol are important sterols present in chickpea oil. Ca, Mg, P and, especially, K are also present in chickpea seeds. Chickpea is a good source of important vitamins such as riboflavin, niacin, thiamin, folate and the vitamin A precursor β-carotene. As with other pulses, chickpea seeds also contain anti-nutritional factors which can be reduced or eliminated by different cooking techniques. Chickpea has several potential health benefits, and, in combination with other pulses and cereals, it could have beneficial effects on some of the important human diseases such as CVD, type 2 diabetes, digestive diseases and some cancers. Overall, chickpea is an important pulse crop with a diverse array of potential nutritional and health benefits.

Modulation of nutrient digestion and absorption is one of the post-ingestion mechanisms that guarantees the best exploitation of food resources, even when they are nutritionally poor or unbalanced, and plays a pivotal role in generalist feeders, which experience an extreme variability in diet composition. Among insects, the larvae of black soldier fly (BSF), Hermetia illucens, can grow on a wide range of feeding substrates with different nutrient content, suggesting that they can set in motion post-ingestion processes to match their nutritional requirements. In the present study we address this issue by investigating how the BSF larval midgut adapts to diets with different nutrient content. Two rearing substrates were compared: a nutritionally balanced diet for dipteran larvae and a nutritionally poor diet that mimics fruit and vegetable waste. Our data show that larval growth performance is only moderately affected by the nutritionally poor diet, while differences in the activity of digestive enzymes, midgut cell morphology, and accumulation of long-term storage molecules can be observed, indicating that diet-dependent adaptation processes in the midgut ensure the exploitation of poor substrates. Midgut transcriptome analysis of larvae reared on the two substrates showed that genes with important functions in digestion and absorption are differentially expressed, confirming the adaptability of this organ.