Carbohydrate Substrates Research Articles

A mild and convenient approach for selective acetonide cleavage involved in carbohydrate synthesis using PPA-SiO2

Here, we report a highly selective, efficient and rapid method for the selective cleavage of primary acetonide using silica supported polyphosphoric acid (PPA-SiO2) for various carbohydrate substrates. Corresponding diols were obtained in good to excellent yields within 30 min using PPA-SiO2. Overall, PPA-SiO2 was found to be a useful catalyst for selective acetonide cleavage in carbohydrate substrates which may expand its utility in organic synthesis.

Применение CRISPR/Cas9-системы редактирования генома в дрожжах Yarrowia lipolytica для инактивации гена YIACL2, кодирующего АТФ-зависимую цитратлиазу

In this study, Y1ACL2 was inactivated by two methods: traditional approach based on homologous recombination and uracil marker and markerless system using CRISPR/Cas9. The efficiency of YIACL2 inactivation using traditional approach was 4% (one ΔYlacl2 strain out of 24 tested transformants) whereas knockout efficiency using CRISPR/Cas9 system was 75% (18 ΔYlacl2 strains out of 24 tested transformants). YIACL2 null mutant strains were not able to utilize citrate as a single carbon source. Growth kinetics was investigated in the media with glucose and acetate as a single carbon source. The fact that ΔYlacl2 is able to grow in the minimal medium with glucose as a single carbon source provides evidence that there is an alternative source of acetyl-CoA on carbohydrate substrates in Y. lipolytica. Yarrowia lipolytica, CRISPR/Cas9 system, ATP citrate lyase, YIACL2. The work was carried out using the equipment of the Unique Scientific Facility of BRC VKPM with technical support of the Centre for Collective Use of NRC «Kurchatov Institute» -GosNIIgenetika and with financial support of Russian Federation (state task No. 595-00003-19 PR) as well as partially supported by grant No. MK-2241.2019.7.

Donor brain stem death and cardiac transplantation causes mitochondrial dyscoupling and oxidative stress

Introduction: Mitochondria are essential for adequate cardiovascular function and are important in the generation of reactive oxygen species (ROS). However, mitochondrial function and ROS post donor brain stem death (BD) and heart transplantation HTx is poorly understood and may contribute to donor heart dysfunction (DHD) and post HTx graft failure (GF). Objectives∖Aims: To examine mitochondrial function and ROS production in hearts both after 24hrs of BSD and post HTx. Methods: Donor sheep underwent BD (BD n=13) or sham instrumentation (SH, n=8) and monitored for 24hrs followed by heart procurement or cold static storage. Orthotopic HTx followed, in 7∖13 BD (BD-Tx) and 4/8 SH donors (SH-Tx). Mitochondrial respirometry was assessed using the Oroboros Oxygraph using carbohydrate (CHO) or fatty acid (FAO) substrates for both ventricles. O2 flux control ratios (FCR) were calculated in LEAK (no ADP) and Complex- I (CI) & II (CII) OXPHOS (with ADP) respiration states. Tissue levels of 3-nitrotyrosine and glutathione:reduced glutathione (GSH:GSSG) ratio were assessed using plate based assays. Results: BD caused a significant increase in LEAK RV proton slip. Both ventricles showed a trend toward higher O2 consumption at CI (known driver of ROS) and a significant decrease in CII respiration for both substrates. Post HTx showed mitochondrial dyscoupling evidenced by a trend toward higher CI O2 consumption, significantly higher RV proton slip, and depressed LV and RV CII respiration. For the LV, GSH:GSSG ratios were significantly lower for BD, BD-Tx and SH-Tx. For the RV, GSH:GSSG ratios were non-significantly depressed post BD both pre and post Tx. Conclusions: We have shown that donor BD and Tx caused significant mitochondrial dyscoupling and higher oxidative stress. Mitochondrial function, particularly CI ROS production may then contribute to DHD and consequently post-HTx GF. Therapeutically targeting mitochondrial ROS could improve patient prognosis.

Sub-stoichiometric reductive etherification of carbohydrate substrates and one-pot protecting group manipulation.

In this study, we report a new reductive etherification procedure for protection of carbohydrate substrates and its application for one-pot preparation of glycosyl building blocks. The reported procedure features the use of polymethylhydrosiloxane (PMHS) as a sub-stoichiometric reducing agent, which prevents the transilylation side reaction and improves the efficiency of the reductive etherification method. Application of the PMHS reductive etherification procedure for one-pot protecting group manipulation are described.

Miscanthus plants processing in fuel, energy, chemical and microbiological industries

The increasing shortage of fossil hydrocarbon fuel dictates the need to search for and develop alternative energy sources, including plant biomass. This paper is devoted to the study of the Miscanthus plants biomass potential and the analysis of technologies of its processing into products targeted at bioenergy, chemistry, and microbiology. Miscanthus is a promising renewable raw material to replace wood raw materials for the production of chemical, fuel, energy, and microbiological industries. Miscanthus is characterised by highly productive (up to 40 tons per one hectare of dry matter) C4-photosynthesis. Dry Miscanthus contains 47.1–49.7% carbon, 5.38–5.92% hydrogen, and 41.4–44.6% oxygen. The mineral composition includes K, Cl, N and S, which influence the processes occurring during biomass combustion. The total amount of extractives per dry substance lies in the range of 0.3–2.2 % for different extraction reagents. Miscanthus has optimal properties as an energy source. Miscanthus × giganteus pellets showed the energy value of about 29 kJ/g. For the bioconversion of plants into bioethanol, it is advisable to carry out simultaneous saccharification and fermentation, thus reducing the duration of process steps and energy costs. Miscanthus cellulose is of high quality and can be used for the synthesis of new products. Further research will focus on the selection of rational parameters for processing miscanthus biomass into products with improved physical and chemical characteristics: bioethanol, pellets, industrial cellulose, bacterial cellulose, carbohydrate substrate.

Identifying adhesive components in a model tunicate.

Tunicates populate a great variety of marine underwater substrates worldwide and represent a significant concern in marine shipping and aquaculture. Adhesives are secreted from the anterior papillae of their swimming larvae, which attach and metamorphose into permanently adhering, filter-feeding adults. We recently described the cellular composition of the sensory adhesive organ of the model tunicate Ciona intestinalis in great detail. Notably, the adhesive secretions of collocytes accumulate at the tip of the organ and contain glycoproteins. Here, we further explore the components of adhesive secretions and have screened for additional specificities that may influence adhesion or cohesion of the Ciona glue, including other carbohydrate moieties, catechols and substrate properties. We found a distinct set of sugar residues in the glue recognized by specific lectins with little overlap to other known marine adhesives. Surprisingly, we also detect catechol residues that likely originate from an adjacent cellular reservoir, the test cells. Furthermore, we provide information on substrate preferences where hydrophobicity outperforms charge in the attachment. Finally, we can influence the settlement process by the addition of hydrophilic heparin. The further analysis of tunicate adhesive strategies should provide a valuable knowledge source in designing physiological adhesives or green antifoulants. This article is part of the theme issue 'Transdisciplinary approaches to the study of adhesion and adhesives in biological systems'.

Bayesian mechanistic modeling of thermodynamically controlled volatile fatty acid, hydrogen and methane production in the bovine rumen

Dynamic modeling of mechanisms driving volatile fatty acid and hydrogen production in the rumen microbial ecosystem contributes to the heuristic prediction of CH4 emissions from dairy cattle into the environment. Existing mathematical rumen models, however, lack the representation of these mechanisms. A dynamic mechanistic model was developed that simulates the thermodynamic control of hydrogen partial pressure (pH2) on volatile fatty acid (VFA) fermentation pathways via the NAD+ to NADH ratio in fermentative microbes, and methanogenesis in the bovine rumen. This model is unique and closely aligns with principles of reaction kinetics and thermodynamics. Model state variables represent ruminal carbohydrate substrates, bacteria and protozoa, methanogens, and gaseous and dissolved fermentation end products. The model was extended with static equations to model the hindgut metabolism. Feed composition and twice daily feeding were used as model inputs. Model parameters were estimated to experimental data using a Bayesian calibration procedure, after which the uncertainty of the parameter distribution on the model output was assessed. The model predicted a marked peak in pH2 after feeding that rapidly declined in time. This peak in pH2 caused a decrease in NAD+ to NADH ratio followed by an increased propionate molar proportion at the expense of acetate molar proportion, and an increase in CH4 production that steadily decreased in time, although the magnitude of increase for CH4 emission was less than for pH2. A global sensitivity analysis indicated that parameters that determine the fractional passage rate and NADH oxidation rate altogether explained 86% of the variation in predicted daily CH4 emission. Model evaluation indicated over-prediction of in vivo CH4 emissions shortly after feeding, whereas under-prediction was indicated at later times. The present rumen fermentation modeling effort uniquely provides the integration of the pH2 controlled NAD+ to NADH ratio for dynamically predicting metabolic pathways that yield VFA, H2 and CH4.

Disentangling the Effects of Temperature, Moisture, and Substrate Availability on Soil CO2 Efflux

AbstractSoil respiration (Rs), the largest carbon emission flux in ecosystems, is usually modeled as an empirically parameterized function of temperature, and sometimes water availability. The likely contribution by other factors, such as carbohydrate substrate supply from photosynthesis, has been recognized, but modeling capacity to use this information is limited. Wavelet transformations of the residuals of a seasonal Q10 temperature response model indicated structure at different temporal scales. We hypothesize that this indicates the lack of explicit representation of relevant processes in the models. Using cross‐spectral analysis, we found that time series of photosynthetically active radiation explained most of the diurnal variation, temperature, explained variability at multiple time scales (diurnal‐synoptic), whereas volumetric soil water content correlated with variability in Rs at scales 15‐30 days. The results suggest that the time domains of influence for different driving variables of Rs are discrete, and largely nonoverlapping, and represent functional relationships between soil biological activity and its constraints. Analysis of phase angles showed that Rs lagged photosynthetically active radiation by 1.5‐3.0 hr. As this time lag was the same in both young and mature trees, with more than fivefold difference in transport distances, we hypothesize that this finding adds to the body of literature that support the pressure‐concentration‐wave model of carbohydrate availability in plants.

A new triazine-thiophene based porous organic polymer as efficient catalyst for the synthesis of chromenes via multicomponent coupling and catalyst support for facile synthesis of HMF from carbohydrates

Functionalized porous organic polymers are very demanding and promising material in heterogeneous catalysis due to their convenience in synthesis and versatility in framework composition. Further, owing to the tunable porosity, high specific surface area, ease of functionalization and chemical stability, this class of porous polymers have huge potential to be employed both as catalysis and catalyst support. Herein, we report N-rich triazine-thiophene based microporous polymer TrzDBTH through the Friedel-Craft alkylation reaction of tripodal building block 2,4,6-tris[4-(bromomethyl)phenyl]-1,3,5-triazine with dibenzothiophene (DBTH) and used it as a heterogeneous base catalyst for one-pot synthesis of biologically important chromene derivatives. Further, sulfonation to this TrzDBTH material yielded sulfonated hyper-crosslinked porous polymer namely STrzDBTH, which can be used as solid acid catalyst for the synthesis of valuable chemical intermediate 5-hydroxymethylfurfural (HMF) from different biomass derived carbohydrates. As a base catalyst, TrzDBTH provides chromene yields from 88 to 93% and its sulfonated derivative STrzDBTH provides the HMF yields from 57 to 97% for different carbohydrate substrates. Both the materials displayed high recycling efficiencies for several catalytic cycles. Thus, these triazine-thiophene based porous organic polymers offer very economical, scalable and recyclable routes for the synthesis chromene derivatives and HMF.

The role of galectin-3 in spermatozoa-zona pellucida binding and its association with fertilization in vitro

Human spermatozoa can fertilize an oocyte only after post-testicular maturation and capacitation. These processes involve dynamic modification and reorganization of the sperm plasma membrane, which allow them to bind to the zona pellucida (ZP) of the oocyte. Defective sperm-ZP binding is one of the major causes of male subfertility. Galectin-3 is a secretory lectin in human seminal plasma well known for its action on cell adhesion. The aim of this study was to determine the role of galectin-3 in spermatozoa-ZP interaction and its association with fertilization rate in clinical assisted reproduction. Our studies revealed that the acrosomal region of ejaculated and capacitated spermatozoa possess strong galectin-3 immunoreactivity, which is much stronger than that of epididymal spermatozoa. Expression of galectin-3 can also be detected on seminal plasma-derived extracellular vesicles (EVs) and can be transferred to the sperm surface. Blocking of sperm surface galectin-3 function by antibody or carbohydrate substrate reduced the ZP-binding capacity of spermatozoa. Purified galectin-3 is capable of binding to ZP, indicating that galectin-3 may serve as a cross-linking bridge between ZP glycans and sperm surface glycoproteins. Galectin-3 levels in seminal plasma-derived EVs were positively associated with fertilization rates. These results suggest that galectin-3 in EVs is transferred to the sperm surface during post-testicular maturation and plays a crucial role in spermatozoa-ZP binding after capacitation. Reduced galectin-3 expression in seminal plasma-derived EVs may be a cause behind a low fertilization rate. Further studies with more clinical samples are required to confirm the relationship between galectin-3 levels and IVF outcomes.

Isolation and characterization of Clostridia from the feces of wild rabbit and swine for hemicellulosic acetone-butanol-ethanol (ABE) production

Isolation and characterization of solventogenic bacteria from animal feces were carried out. Ten samples were collected continually for 5 d from the feces of wild rabbit and wild swine in Jawaharlal Nehru Zoological Park, Hyderabad, India. Ten acetone-positive strains were selected for evaluation of their phenotypic and physiological characteristics. Two potential solvent-producing cultures were selected for 16S rRNA gene analysis. The culture isolated from the wild rabbit feces exhibited 97.3% similarity with Clostridium beijerinckii 8052, and the culture isolated from the wild swine feces exhibited 93.8% similarity with Clostridium saccharoperbutylacetonicum NI-4(HMT). The isolated strains utilized a wide range of carbohydrate substrates including glucose, fructose, maltose, xylose, arabinose, and glycerol. The major fermentation products from glucose, xylose, and arabinose were acetone, butanol, and ethanol (ABE). The total ABE concentration produced by strain YVU1 was 13.1 g/L from arabinose, 16.3 g/L from xylose, and 20.6 g/L from glucose. Strain YVU2 produced a total ABE concentration of 16.5 g/L, 18.3 g/L, and 22.4 g/L from arabinose, xylose, and glucose, respectively.

Diversity and Functional Properties of Lactic Acid Bacteria Isolated From Wild Fruits and Flowers Present in Northern Argentina.

Lactic acid bacteria (LAB) are capable of converting carbohydrate substrates into organic acids (mainly lactic acid) and producing a wide range of metabolites. Due to their interesting beneficial properties, LAB are widely used as starter cultures, as probiotics, and as microbial cell factories. Exploring LAB present in unknown niches may lead to the isolation of unique species or strains with relevant technological properties. Autochthonous rather than allochthonous starter cultures are preferred in the current industry of fermented food products, due to better adaptation and performance of autochthonous strains to the matrix they originate from. In this work, the lactic microbiota of eight different wild tropical types of fruits and four types of flowers were studied. The ability of the isolated strains to produce metabolites of interest to the food industry was evaluated. The presence of 21 species belonging to the genera Enterococcus, Fructobacillus, Lactobacillus, Lactococcus, Leuconostoc, and Weissella was evidenced by using culture-dependent techniques. The isolated LAB corresponded to 95 genotypically differentiated strains by applying rep-PCR and sequencing of the 16S rRNA gene; subsequently, representative strains of the different isolated species were studied for technological properties, such as fast growth rate and acidifying capacity; pectinolytic and cinnamoyl esterase activities, and absence of biogenic amine biosynthesis. Additionally, the strains' capacity to produce ethyl esters as well as mannitol was evaluated. The isolated fruit- and flower-origin LAB displayed functional properties that validate their potential use in the manufacture of fermented fruit-based products setting the background for the design of novel functional foods.

Biobutanol production from cassava waste residue using Clostridium sp. AS3 in batch culture fermentation

Tapioca starch factories generate a large amount of wastewater and solid waste, which take several steps of waste treatment to manage. The solid waste contains high level of starchy lignocellulosic materials, especially cassava pulp (CP). The cassava waste residue (CWR) is a potentially promising substrate for the production of biobutanol. Clostridium sp. strain AS3 isolated from a cassava waste processing industrial site utilised a wide range of carbohydrate substrates in a pre-optimised CWR medium. The biobutanol production capacity of the strain was assessed under test conditions of 5% butanol tolerance, pH 6.3, CWR 60 g L−1, pre-treatment acid hydrolysis, inoculum volume 5% v/v, temperature 33 °C, flow rate 0.8 L/L/min, agitation 85 rpm and incubation time 72 h, in serum bottles and also in 5-L reactors for possible scale-up. The results showed that the strain produced 10.5 g L−1 of acetone–butanol–ethanol at a 1:4:11 ratio (v/v/v) from CWR. In subsequent scale-up, the strain produced 0.11 g/L/h of biobutanol that was 70% total solvents produced from CWR. Overall, the batch study showed promising results that the CWR was potentially valorised by solventogenic Clostridium sp. AS3, and this strain has the potential for continuous biobutanol production.

Molecular Mechanisms of Oxygen Activation and Hydrogen Peroxide Formation in Lytic Polysaccharide Monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes for the degradation of recalcitrant polysaccharides such as chitin and cellulose. Unlike classical hydrolytic enzymes (cellulases), LPMOs catalyze the cleavage of the glycosidic bond via an oxidative mechanism using oxygen and a reductant. The full enzymatic molecular mechanisms, starting from the initial electron transfer from a reductant to oxygen activation and hydrogen peroxide formation, are not yet understood. Using quantum mechanics/molecular mechanics (QM/MM) metadynamics simulations, we have uncovered the oxygen activation mechanisms by LPMO in the presence of ascorbic acid, one of the most-used reductants in LPMOs assays. Our simulations capture the sequential formation of Cu(II)-O2– and Cu(II)-OOH– intermediates via facile H atom abstraction from ascorbate. By investigating all the possible reaction pathways from the Cu(II)–OOH– intermediate, we ruled out Cu(II)-O• – formation via direct O–O cleavage of Cu(II)-OOH–. Meanwhile, we identified a possible pathway in which the proximal O atom of Cu(II)–OOH– abstracts a hydrogen atom from ascorbate, leading to Cu(I) and H2O2. The in-situ-generated H2O2 either converts to LPMO-Cu(II)-O• – via a homolytic reaction, or diffuses into the bulk water in an uncoupled pathway. The competition of these two pathways is strongly dependent on the binding of the carbohydrate substrate, which plays a role in barricading the in-situ-generated H2O2 molecule, preventing its diffusion from the active site into the bulk water. Based on the present results, we propose a catalytic cycle of LPMOs that is consistent with the experimental information available. In particular, it explains the enigmatic substrate dependence of the reactivity of the LPMO with H2O2.

Control of Cardiac Mitochondrial Fuel Selection by Calcium

Calcium ion concentration modulates the function of several mitochondrial enzymes. Specifically, the kinetic operations of the decarboxylating dehydrogenases pyruvate dehydrogenase, isocitrate dehydrogenase, alpha‐ketoglutarate dehydrogenase are all affected by [Ca2+]. Previous studies have shown that, despite its ability to affect the function of specific dehydrogenases, [Ca2+] does not substantially alter mitochondrial ATP synthesis in vitro or in vivo in the heart. We hypothesize that, rather than contributing to respiratory control, [Ca2+] plays a role in contributing to fuel selection. Specifically, cardiac mitochondria are able to use different primary carbon substrates (carbohydrates, fatty acids, and ketones) to synthesize ATP aerobically in the living cells. To determine if and how [Ca2+] affects the relative use of carbohydrates versus fatty acids in vitro we measured oxygen consumption and TCA cycle intermediate concentrations in suspensions of cardiac mitochondria with different combinations of pyruvate and palmitoyl‐L‐carnitine in the media at various [Ca2+] and ADP infusion rates. Stoichiometric analysis of the data reveals that when both fatty acid and carbohydrate substrates are available, fuel selection is sensitive to both the rate of ADP infusion and to the calcium concentration. With increasing both the rate of oxidative ATP synthesis and [Ca2+], the fuel utilization ratio shifts to increased fractional consumption of pyruvate. Thus, our results indicate that the effects of ATP synthesis rate and [Ca2+] are interdependent.Support or Funding InformationThis work supported by NIH grants HL122199 and T32GM008322This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Structural Studies of a Novel Glucan Phosphatase from the Red Alga Cyanidioschyzon Merolae

BackgroundGlucan phosphatases are a unique subset of the Dual Specificity Phosphatase (DSP) family that binds and dephosphorylates carbohydrate substrates. They are found in widely divergent organisms ranging from extremophilic single‐cell red algae to humans and act on different carbohydrate (starch or glycogen) substrates. Red algae, Cyanidioschyzon merolae (Cm) possesses a single glucan phosphatase with three structural domains which are different in family and organization compared to other known glucan phosphatase family members. Further, bioinformatics analysis suggested that it is most similar to a vertebrate glucan phosphatase rather than plant glucan phosphatases. This is intriguing, since its endogenous substrate is starch rather than glycogen.HypothesisCm‐laforin is uniquely stable and active glucan phosphatase and can be an important partner with other catabolic enzymes in the process of starch metabolism.ObjectiveExplore the structural basis for Cm‐laforin's function as a novel glucan phosphatase.MethodsWe expressed and purified the full, tandem, and isolated carbohydrate binding (CBM) and phosphatase (DSP) domains of Cm‐laforin to study their thermal stability, phosphatase function, and domain coupling. We utilized para‐nitrophenylphosphate (p‐NPP) substrate for generic and amylopectin in malachite green assay for specific phosphatase activities determination. Differential scanning fluorimetry (DSF) and starch binding assays were utilized for glucan binding studies. The oligomeric state of laforin was determined by Size Exclusion Chromatography with Multi‐Angle static Light Scattering (SEC‐MALS). X‐ray crystallography was utilized for structural studies, which allowed structure‐guided mutagenesis.ResultsCm‐laforin is found to be a stable and highly active glucan phosphatase. The full three‐domain protein was found to be highly active and have maximum phosphatase activity at 55°C. Further, it possesses 2‐fold higher specific glucan phosphatase activity compared to vertebrate laforin orthologs. Utilizing the tools of structural biology, we explored the importance of different amino acids of the DSP domain required for its stability. We determined the structure of the DSP domain at 1.5Å and found that Cm‐laforin utilizes a significant number of unique electrostatic interactions centered on D89 to maintain its significantly higher stability. We also found that CBM1 and DSP domain can be expressed and function in isolation, but CBM1 and linker region between CBM2 and DSP are required for the proper folding and stability of CBM2. We further demonstrate that Cm‐laforin exists as a monomer, and that the tandem CBM domains are required for high affinity glucan binding and enzymatic coupling during starch degradation.ConclusionCm‐laforin is enzymatically highly active and stable phosphatase with unique structural and functional features that underlie its role as a specific glucan phosphatase.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Discovery of novel carbohydrate-active enzymes through the rational exploration of the protein sequences space

Over the last two decades, the number of gene/protein sequences gleaned from sequencing projects of individual genomes and environmental DNA has grown exponentially. Only a tiny fraction of these predicted proteins has been experimentally characterized, and the function of most proteins remains hypothetical or only predicted based on sequence similarity. Despite the development of postgenomic methods, such as transcriptomics, proteomics, and metabolomics, the assignment of function to protein sequences remains one of the main challenges in modern biology. As in all classes of proteins, the growing number of predicted carbohydrate-active enzymes (CAZymes) has not been accompanied by a systematic and accurate attribution of function. Taking advantage of the CAZy database, which groups CAZymes into families and subfamilies based on amino acid similarities, we recombinantly produced 564 proteins selected from subfamilies without any biochemically characterized representatives, from distant relatives of characterized enzymes and from nonclassified proteins that show little similarity with known CAZymes. Screening these proteins for activity on a wide collection of carbohydrate substrates led to the discovery of 13 CAZyme families (two of which were also discovered by others during the course of our work), revealed three previously unknown substrate specificities, and assigned a function to 25 subfamilies.

Effects of nitrogen supply on flavonol glycoside biosynthesis and accumulation in tea leaves (Camellia sinensis)

Widely distributed in tea plants, the flavonoid flavonol and its glycosylated derivatives have important roles in determining tea quality. However, the biosynthesis and accumulation of these compounds has not been fully studied, especially in response to nitrogen (N) supply. In the present study, ‘Longjing 43’ potted tea seedlings were subjected to N deficiency (0g/pot), normal N (4g/pot) or excess N (16g/pot). Quantitative analyses using Ultra Performance Liquid Chromatography-Triple Quadrupole Mass Spectrometry (UPLC-QqQ-MS/MS) revealed that most flavonol glycosides (e.g., Quercetin-3-glucoside, Kaempferol-3-rgalactoside and Kaempferol-3-glucosyl-rhamnsoyl-glucoside) accumulated to the highest levels when treated with normal N. Results from metabolomics using Gas Chromatography-Mass Spectrometer (GC-MS) suggested that the levels of carbohydrate substrates of flavonol glycosides (e.g., sucrose, sucrose-6-phosphate, D-fructose 1,6-bisphosphate and glucose-1-phosphate) were positively correlated with flavonol glycoside content in response to N availability. Furthermore, Quantitative Real-time PCR analysis of 28 genes confirmed that genes related to flavonoid (e.g., flavonol synthase 1, flavonol 3-O-galactosyltransferase) and carbohydrate (e.g., sucrose phosphate synthase, sucrose synthase and glucokinase) metabolism have important roles in regulating the biosynthesis and accumulation of flavonol glycosides. Collectively, our results suggest that normal N levels promote the biosynthesis of flavonol glycosides through gene regulation and the accumulation of substrate carbohydrates, while abnormal N availability has inhibitory effects, especially excess N.

Butyrivibrio hungateiMB2003 Competes Effectively for Soluble Sugars Released byButyrivibrio proteoclasticusB316Tduring Growth on Xylan or Pectin

Rumen bacterial species belonging to the genus Butyrivibrio are important degraders of plant polysaccharides, particularly hemicelluloses (arabinoxylans) and pectin. Currently, four species are recognized; they have very similar substrate utilization profiles, but little is known about how these microorganisms are able to coexist in the rumen. To investigate this question, Butyrivibrio hungatei MB2003 and Butyrivibrio proteoclasticus B316T were grown alone or in coculture on xylan or pectin, and their growth, release of sugars, fermentation end products, and transcriptomes were examined. In monocultures, B316T was able to grow well on xylan and pectin, while MB2003 was unable to utilize either of these insoluble substrates to support significant growth. Cocultures of B316T grown with MB2003 revealed that MB2003 showed growth almost equivalent to that of B316T when either xylan or pectin was supplied as the substrate. The effect of coculture on the transcriptomes of B316T and MB2003 was assessed; B316T transcription was largely unaffected by the presence of MB2003, but MB2003 expressed a wide range of genes encoding proteins for carbohydrate degradation, central metabolism, oligosaccharide transport, and substrate assimilation, in order to compete with B316T for the released sugars. These results suggest that B316T has a role as an initiator of primary solubilization of xylan and pectin, while MB2003 competes effectively for the released soluble sugars to enable its growth and maintenance in the rumen.IMPORTANCE Feeding a future global population of 9 billion people and climate change are the primary challenges facing agriculture today. Ruminant livestock are important food-producing animals, and maximizing their productivity requires an understanding of their digestive systems and the roles played by rumen microbes in plant polysaccharide degradation. Butyrivibrio species are a phylogenetically diverse group of bacteria and are commonly found in the rumen, where they are a substantial source of polysaccharide-degrading enzymes for the depolymerization of lignocellulosic material. Our findings suggest that closely related species of Butyrivibrio have developed unique strategies for the degradation of plant fiber and the subsequent assimilation of carbohydrates in order to coexist in the competitive rumen environment. The identification of genes expressed during these competitive interactions gives further insight into the enzymatic machinery used by these bacteria as they degrade the xylan and pectin components of plant fiber.

Physical Activity and Its Impact on Diabetes

The current wave of the diabetes epidemic has become a global public health issue. Multiple strategies have been studied in trying to curb the menace and promising among these is lifestyle intervention which includes regular physical activity. During physical activity there is an accelerated utilization of substrates particularly stored triglycerides by the muscles thereby improving insulin sensitivity and this is valuable in the prevention of diabetes. On prolonged exercise blood glucose is derived from the breakdown of glycogen and from non carbohydrate substrates via gluconeogenesis. Exercise has a positive effect on diabetes co-morbidities e.g. obesity, hypertension, dyslipidemia as well as all-cause mortality. Regular physical activity mediates molecular events in the various pathways of diabetic complications like exerting anti-inflammatory effects to combat the chronic subclinical inflammation, fortifying the body antioxidant defense systems and reducing the formation and effects of advanced glycation end-products. The maximum health benefits of exercise are obtainable when done regularly and guidelines are available for the general public and individuals with diabetes on how to achieve these benefits. Finally, the emphasis on the importance of physical activity does not exclude the principle that patients with diabetic complications should seek expert counseling before embarking on an exercise program because some activities might be risky for this group of patients.