Sucrose Uptake Research Articles

Study on Mass Transfer and Physicochemical Properties of Avocado During Osmotic Dehydration

A previous study found that mass transfer occurring during the osmotic dehydration (OD) process. This phenomena is possible causing modification in physicochemical properties; therefore, understanding the OD effect on quality attributes is important. This research aimed to study the impact of sucrose concentration and immersion time on the kinetics of mass transfer and the physicochemical properties of avocado (Persea americana var. Miki) during the OD process. The samples were immersed in a sucrose solution (45, 55 and 65 ° Brix) for up to 300 min, then the mass transfer parameter and physicochemical properties were evaluated. The results showed that the highest rate of water removal (WR) was –0.037 g.g–1.h–1 using the 55 ° Brix solution, which also led to an increase in the soluble solid gain (SSG) value to 0.0126 g.g–1.h–1, while the highest WR/SSG ratio was obtained after 180 min of immersion using 65 ° Brix (2.451). A 2nd-order polynomial model accurately describes avocado mass transfer kinetics and their relationship to physicochemical properties. The model showed the best adjustment for WR, SSG, and weight loss (WL), as pointed out by R2 = 0.9671, 0.8968 and 0.9573, respectively. WR has a negative effect on firmness (R2 = 0.9278), vitamin C (R2 = 0.9349), and total fat (R2 = 0.8954). The uptake of sucrose correlated with decrease of vitamin C (R2 = 0.9391), whereas WL also affected the loss of total fat (R2 = 0.8288). The OD process modified the chroma of the avocado by reducing lightness, increasing greenness and yellowness, and yet protecting the flesh from the browning effect. Overall, this study provides insights into the relationship between mass transfer and physicochemical properties, and it is useful to predict the final condition of the osmo-avocado before subsequent processes. HIGHLIGHTS Immersing the avocado in a 55 ° Brix solution resulted in the highest rate of water removal (WR) and soluble solid gain (SSG). The highest ratio of WR and soluble solid gain (WR/SSG) was obtained at 180 min of immersion using a 65 ° Brix solution. The 2nd-order polynomial model fits the mass transfer parameters (WR, SSG, and weight loss/WL). WR contributed to a softened texture, color contrast, degradation of vitamin C, and total fat content. The combination of mass transfer and physicochemical property models is useful for designing the OD process and predicting the final condition of osmo-avocado. GRAPHICAL ABSTRACT

Sucrose as an electron source for cofactor regeneration in recombinant Escherichia coli expressing invertase and a Baeyer Villiger monooxygenase

BackgroundThe large-scale biocatalytic application of oxidoreductases requires systems for a cost-effective and efficient regeneration of redox cofactors. These represent the major bottleneck for industrial bioproduction and an important cost factor. In this work, co-expression of the genes of invertase and a Baeyer–Villiger monooxygenase from Burkholderia xenovorans to E. coli W ΔcscR and E. coli BL21 (DE3) enabled efficient biotransformation of cyclohexanone to the polymer precursor, ε-caprolactone using sucrose as electron source for regeneration of redox cofactors, at rates comparable to glucose. E. coli W ΔcscR has a native csc regulon enabling sucrose utilization and is deregulated via deletion of the repressor gene (cscR), thus enabling sucrose uptake even at concentrations below 6 mM (2 g L−1). On the other hand, E. coli BL21 (DE3), which is widely used as an expression host does not contain a csc regulon.ResultsHerein, we show a proof of concept where the co-expression of invertase for both E. coli hosts was sufficient for efficient sucrose utilization to sustain cofactor regeneration in the Baeyer–Villiger oxidation of cyclohexanone. Using E. coli W ΔcscR, a specific activity of 37 U gDCW−1 was obtained, demonstrating the suitability of the strain for recombinant gene co-expression and subsequent whole-cell biotransformation. In addition, the same co-expression cassette was transferred and investigated with E. coli BL21 (DE3), which showed a specific activity of 17 U gDCW− 1. Finally, biotransformation using photosynthetically-derived sucrose from Synechocystis S02 with E. coli W ΔcscR expressing BVMO showed complete conversion of cyclohexanone after 3 h, especially with the strain expressing the invertase gene in the periplasm.ConclusionsResults show that sucrose can be an alternative electron source to drive whole-cell biotransformations in recombinant E. coli strains opening novel strategies for sustainable chemical production.

Reversible assembly and disassembly of V-ATPase during the lysosome regeneration cycle.

Regulation of the luminal pH of late endocytic compartments in continuously fed mammalian cells is poorly understood. Using normal rat kidney fibroblasts, we investigated the reversible assembly/disassembly of the proton pumping V-ATPase when endolysosomes are formed by kissing and fusion of late endosomes with lysosomes and during the subsequent reformation of lysosomes. We took advantage of previous work showing that sucrosomes formed by the uptake of sucrose are swollen endolysosomes from which lysosomes are reformed after uptake of invertase. Using confocal microscopy and subcellular fractionation of NRK cells stably expressing fluorescently tagged proteins, we found net recruitment of the V1 subcomplex during sucrosome formation and loss during lysosome reformation, with a similar time course to RAB7a loss. Addition of invertase did not alter mTORC1 signalling, suggesting that the regulation of reversible V-ATPase assembly/disassembly in continuously fed cells differs from that in cells subject to amino acid depletion/refeeding. Using live cell microscopy, we demonstrated recruitment of a fluorescently tagged V1 subunit during endolysosome formation and a dynamic equilibrium and rapid exchange between the cytosolic and membrane bound pools of this subunit. We conclude that reversible V-ATPase assembly/disassembly plays a key role in regulating endolysosomal/lysosomal pH in continuously fed cells.

Sugar beet PMT5a and STP13 carriers suitable for proton-driven plasma membrane sucrose and glucose import in taproots.

Sugar beet (Beta vulgaris) is the major sugar-producing crop in Europe and Northern America, as the taproot stores sucrose at a concentration of around 20%. Genome sequence analysis together with biochemical and electrophysiological approaches led to the identification and characterization of the TST sucrose transporter driving vacuolar sugar accumulation in the taproot. However, the sugar transporters mediating sucrose uptake across the plasma membrane of taproot parenchyma cells remained unknown. As with glucose, sucrose stimulation of taproot parenchyma cells caused inward proton fluxes and plasma membrane depolarization, indicating a sugar/proton symport mechanism. To decipher the nature of the corresponding proton-driven sugar transporters, we performed taproot transcriptomic profiling and identified the cold-induced PMT5a and STP13 transporters. When expressed in Xenopus laevis oocytes, BvPMT5a was characterized as a voltage- and H+-driven low-affinity glucose transporter, which does not transport sucrose. In contrast, BvSTP13 operated as a high-affinity H+/sugar symporter, transporting glucose better than sucrose, and being more cold-tolerant than BvPMT5a. Modeling of the BvSTP13 structure with bound mono- and disaccharides suggests plasticity of the binding cleft to accommodate the different saccharides. The identification of BvPMT5a and BvSTP13 as taproot sugar transporters could improve breeding of sugar beet to provide a sustainable energy crop.

Higher Brain Uptake of Gentamicin and Ceftazidime under Isoflurane Anesthesia Compared to Ketamine/Xylazine.

We have recently shown that the volatile anesthetics isoflurane and sevoflurane acutely enhance the brain uptake of the hydrophilic markers sucrose and mannitol about two-fold from an awake condition, while the combined injection of the anesthetic agents ketamine and xylazine has no effect. The present study investigated two small-molecule hydrophilic drugs with potential neurotoxicity, the antibiotic agents ceftazidime and gentamicin. Transport studies using an in vitro blood-brain barrier (BBB) model, a monolayer of induced pluripotent stem cell-derived human brain microvascular endothelial cells seeded on Transwells, and LC-MS/MS analysis demonstrated the low permeability of both drugs in the range of sucrose, with permeability coefficients of 6.62 × 10-7 ± 2.34 × 10-7 cm/s for ceftazidime and 7.38 × 10-7 ± 2.29 × 10-7 cm/s for gentamicin. In vivo brain uptake studies of ceftazidime or gentamicin after IV doses of 25 mg/kg were performed in groups of 5-6 mice anesthetized at typical doses for surgical procedures with either isoflurane (1.5-2% v/v) or ketamine/xylazine (100:10 mg/kg I.P.). The brain uptake clearance, Kin, for ceftazidime increased from 0.033 ± 0.003 μL min-1 g-1 in the ketamine/xylazine group to 0.057 ± 0.006 μL min-1 g-1 in the isoflurane group (p = 0.0001), and from 0.052 ± 0.016 μL min-1 g-1 to 0.101 ± 0.034 μL min-1 g-1 (p = 0.0005) for gentamicin. We did not test the dose dependency of the uptake, because neither ceftazidime nor gentamicin are known substrates of any active uptake or efflux transporters at the BBB. In conclusion, the present study extends our previous findings with permeability markers and suggests that inhalational anesthetic isoflurane increases the BBB permeability of hydrophilic small-molecule endobiotics or xenobiotics when compared to the injection of ketamine/xylazine. This may be of clinical relevance in the case of potential neurotoxic substances.

Differential metabolic reprogramming in developing soybean embryos in response to nutritional conditions and abscisic acid.

Seed storage compound deposition is influenced by both maternal and filial tissues. Within this framework, we analyzed strategies that operate during the development and filling of soybean embryos, using in vitro culture systems combined with metabolomics and proteomics approaches. The carbon:nitrogen ratio (C:N) of the maternal supply and the hormone abscisic acid (ABA) are specific and interacting signals inducing differential metabolic reprogrammings linked to changes in the accumulation of storage macromolecules like proteins or oils. Differences in the abundance of sugars, amino acids, enzymes, transporters, transcription factors, and proteins involved in signaling were detected. Embryos adapted to the nutritional status by enhancing the metabolism of both carbon and nitrogen under lower C:N ratio condition or only carbon under higher C:N ratio condition. ABA turned off multiple pathways especially in high availability of amino acids, prioritizing the storage compounds biosynthesis. Common responses induced by ABA involved increased sucrose uptake (to increase the sink force) and oleosin (oil body structural component) accumulation. In turn, ABA differentially promoted protein degradation under lower nitrogen supply in order to sustain the metabolic demands. Further, the operation of a citrate shuttle was suggested by transcript quantification and enzymatic activity measurements. The results obtained are useful to help define biotechnological tools and technological approaches to improve oil and protein yields, with direct impact on human and animal nutrition as well as in green chemistry.

Microglial stimulation triggered by intranasal lipopolysaccharide administration produces antidepressant-like effect through ERK1/2-mediated BDNF synthesis in the hippocampus

We recently reported that reversing the chronic stress-induced decline of microglia in the dentate gyrus (DG) of the hippocampus by intraperitoneal injection of a low dose of lipopolysaccharide (LPS) ameliorated depression-like behavior in chronically stressed mice. In this study, we found that a single intranasal administration of LPS dose-dependently improved depression-like behavior in mice treated with chronic unpredictable stress (CUS), as evidenced by the reduction of immobility time in the tail suspension test (TST) and forced swimming test (FST) and by the increase of sucrose uptake in the sucrose preference test (SPT). The antidepressant effects of intranasal administration of LPS could be abolished by inhibition of brain-derived neurotrophic factor (BDNF) signaling by infusion of an anti-BDNF antibody, by knock-in of the mutant BDNF Val68Met allele, or by the BDNF receptor antagonist K252a. In addition, intranasal administration of LPS was found to exert antidepressant effects in a BDNF-dependent manner via promotion of BDNF synthesis mediated by extracellular signal-regulated kinase 1/2 (ERK1/2) signaling but not protein kinase B (Akt)-mammalian target of rapamycin (mTOR) signaling in DG. Inhibition of microglia by minocycline or depletion of microglia by PLX3397 was able to abolish the reversal effect of intranasal LPS administration on CUS-induced depression-like behaviors as well as the CUS-induced decrease in phospho-ERK1/2 and BDNF protein levels in DG. These results demonstrate that stimulation of hippocampal microglia by intranasal LPS administration can induce antidepressant effects via ERK1/2-dependent synthesis of BDNF protein, providing hope for the development of new strategies for the treatment of depression.

Carbon Fluxes in Potato (Solanum tuberosum) Remain Stable in Cell Cultures Exposed to Nutritional Phosphate Deficiency.

Nutritional phosphate deficiency is a major limitation to plant growth. Here, we monitored fluxes in pathways supporting respiratory metabolism in potato (Solanum tuberosum) cell cultures growing in control or limiting phosphate conditions. Sugar uptake was quantified using [U-14C]sucrose as precursor. Carbohydrate degradation through glycolysis and respiratory pathways was estimated using the catabolism of [U-14C]sucrose to 14CO2. Anaplerotic carbon flux was assessed by labeling with NaH14CO3. The data showed that these metabolic fluxes displayed distinct patterns over culture time. However, phosphate depletion had relatively little impact on the various fluxes. Sucrose uptake was higher during the first six days of culture, followed by a decline, which was steeper in Pi-sufficient cells. Anaplerotic pathway flux was more important at day three and decreased thereafter. In contrast, the flux between sucrose and CO2 was at a maximum in the mid-log phase of the culture, with a peak at Day 6. Metabolization of [U-14C]sucrose into neutral, basic and acidic fractions was also unaffected by phosphate nutrition. Hence, the well-documented changes in central metabolism enzymes activities in response to Pi deficiency do not drastically modify metabolic fluxes, but rather result in the maintenance of the carbon fluxes that support respiration.

Pea protein isolate and inulin as plant‐based biomacromolecules for reduction of sugar uptake in osmotic dehydration

AbstractConcerns have been raised about the potential health risks associated with elevated sugar levels in food products of osmotic dehydration (OD). To address this issue, a new strategy was proposed and validated in this study. Pea protein isolate (PPI) and inulin were introduced into osmotic solutions to reduce sucrose uptake in apple slices. Apple samples were subjected to OD (40%wt sucrose) at 40°C with different concentrations of PPI or inulin. Three parameters, including performance ratio (Pr), dehydration efficiency index (DEI), and sucrose gain rate were used to evaluate the effect of PPI and inulin addition on reducing sucrose uptake. The result showed that sucrose content in apple tissues after OD for 4 h was 51.79 mg/100 mg dry basis when treated by a sucrose solution alone, but was reduced to 43.43 mg/100 mg dry basis and 46.12 mg/100 mg dry basis when 1.5%wt inulin and 3% wt PPI was added, respectively.Practical applicationsThe use of low‐temperature and phase‐change‐free osmotic dehydration methods for the preservation of fruits is advantageous due to low energy consumption, better quality retention, and ease of operation. It has been used to produce intermediate moisture foods or as a pretreatment for other drying methods. However, the most widely used osmotic agents often contain high calories because carbohydrates have become the most popular osmotic agents. This has promoted the exploration of natural methods to reduce sugar uptake while maintaining the drying characteristic of osmotic dehydration. This study was undertaken to investigate the use of two biomacromolecules, that is, pea protein isolate and inulin as a simple yet effective means for achieving sugar reduction. The finding of this work has demonstrated that both methods can significantly reduce sucrose content in dried apple slices, thus confirming the potential for practical applications.

Intranasal administration of lipopolysaccharide reverses chronic stress-induced depression-like behavior in mice by microglial stimulation

We recently reported that intraperitoneal injection of a low dose of lipopolysaccharide (LPS) reversed depression-like behavior in mice induced by chronic stress by stimulating microglia in the hippocampus. In this study, we found that a single intranasal administration of LPS at a dose of 5 or 10 μg/mouse, but not at a dose of 1 μg/mouse, rapidly reversed depression-like behavior in mice stimulated with chronic unpredictable stress (CUS). In the time-dependent experiment, a single intranasal administration of LPS (10 μg/mouse) reversed CUS-induced depression-like behavior in mice 5 and 8 h but not 3 h after drug administration. The antidepressant effect of a single intranasal LPS administration (10 μg/mouse) lasted at least 10 days and disappeared 14 days after administration. Fourteen days after the first intranasal LPS administration, a second intranasal LPS administration (10 μg/mouse) still reversed the increased immobility time in TST and FST and the decreased sucrose uptake in SPT in CUS mice, which again exhibited depression-like behaviors 5 h after LPS administration. The antidepressant effect of intranasal LPS administration was dependent on microglial activation, because inhibition of microglia by pretreatment with minocycline (40 mg/kg) or depletion of microglia by pretreatment with PLX3397 (290 mg/kg) prevented the antidepressant effect of intranasal LPS administration in CUS mice. These results suggest that stimulation of the microglia-mediated innate immune response by intranasal administration of LPS can produce rapid and sustained antidepressant effects in animals under chronic stress conditions.

Structure and sucrose binding mechanism of the plant SUC1 sucrose transporter

Sucrose import from photosynthetic tissues into the phloem is mediated by transporters from the low-affinity sucrose transporter family (SUC/SUT family). Furthermore, sucrose redistribution to other tissues is driven by phloem sap movement, the product of high turgor pressure created by this import activity. Additionally, sink organs such as fruits, cereals and seeds that accumulate high concentrations of sugar also depend on this active transport of sucrose. Here we present the structure of the sucrose–proton symporter, Arabidopsis thaliana SUC1, in an outward open conformation at 2.7 Å resolution, together with molecular dynamics simulations and biochemical characterization. We identify the key acidic residue required for proton-driven sucrose uptake and describe how protonation and sucrose binding are strongly coupled. Sucrose binding is a two-step process, with initial recognition mediated by the glucosyl moiety binding directly to the key acidic residue in a stringent pH-dependent manner. Our results explain how low-affinity sucrose transport is achieved in plants, and pinpoint a range of SUC binders that help define selectivity. Our data demonstrate a new mode for proton-driven symport with links to cation-driven symport and provide a broad model for general low-affinity transport in highly enriched substrate environments.

Effects of Post-Anthesis High-Temperature Stress on Carbon Partitioning and Starch Biosynthesis in a Spring Wheat (Triticum aestivum L.) Adapted to Moderate Growth Temperatures.

This study investigates carbon partitioning in the developing endosperm of a European variety of spring wheat subjected to moderately elevated daytime temperatures (27°C/16°C d/night) from anthesis to grain maturity. Elevated daytime temperatures caused significant reductions in both fresh and dry weights and reduced the starch content of harvested grains compared to plants grown under a 20°C/16°C d/night regimen. Accelerated grain development caused by elevated temperatures was accounted for by representing plant development as thermal time (°C DPA). We examined the effects of high-temperature stress (HTS) on the uptake and partitioning of [U-14C]-sucrose supplied to isolated endosperms. HTS caused reduced sucrose uptake into developing endosperms from the second major grain-filling stage (approximately 260°C DPA) up to maturity. Enzymes involved in sucrose metabolism were unaffected by HTS, whereas key enzyme activities involved in endosperm starch deposition such as ADP-glucose pyrophosphorylase and soluble isoforms of starch synthase were sensitive to HTS throughout grain development. HTS caused a decrease in other major carbon sinks such as evolved CO2, ethanol-soluble material, cell walls and protein. Despite reductions in the labeling of carbon pools caused by HTS, the relative proportions of sucrose taken up by endosperm cells allocated to each cellular pool remain unchanged, except for evolved CO2, which increased under HTS and may reflect enhanced respiratory activity. The results of this study show that moderate temperature increases can cause significant yield reductions in some temperate wheat cultivars chiefly through three effects: reduced sucrose uptake by the endosperm, reduced starch synthesis and increased partitioning of carbon into evolved CO2.

Pericarp starch metabolism is associated with caryopsis development and endosperm starch accumulation in common wheat

The wheat pericarp is the main component of the caryopsis at the early development stage and ultimately converts into a tissue that covers the mature caryopsis. A large number of starch granules are accumulated in the pericarp, but the production of and the role of starch granules in caryopsis development remain- elusive. In the present study, the relationship between accumulated starch granules and starch metabolism-related genes in wheat pericarp was investigated using paraffin section observations, expression analysis, and mutant analysis. Starch synthesis is initiated before anthesis and is dependent on a sucrose uptake and conversion system similar to that in the endosperm. TaPTST2 is required to initiate the production of pericarp starch granules. Pericarp starch granules gradually disappeared at the filling stage with high expression levels of genes encoding β-amylase, sucrose-phosphate synthase, and sucrose-phosphate phosphatase. As a maternal tissue adjacent to the endosperm and embryo, the pericarp plays a temporary reservoir for excess nutrients delivered into the caryopsis during the early development stage and exported at the filling stage. The pericarp contributes to the development of the endosperm and embryo as well as the accumulation of endosperm starch. The metabolism of pericarp starch may affect the weight of the wheat caryopsis.

Sugar concentrations and expression of SUTs suggest active phloem loading in tall trees of Fagus sylvatica and Quercus robur.

Phloem loading and sugar distribution are key steps for carbon partitioning in herbaceous and woody species. Although the phloem loading mechanisms in herbs are well studied, less is known for trees. It was shown for saplings of Fagus sylvatica L. and Quercus robur L. that the sucrose concentration in the phloem sap was higher than in the mesophyll cells, which suggests that phloem loading of sucrose involves active steps. However, the question remains whether this also applies for tall trees. To approach this question, tissue-specific sugar and starch contents of small and tall trees of F. sylvatica and Q. robur as well as the sugar concentration in the subcellular compartments of mesophyll cells were examined. Moreover, sucrose uptake transporters (SUTs) were analyzed by heterology expression in yeast and the tissue-specific expressions of SUTs were investigated. Sugar content in leaves of the canopy (11 and 26m height) was up to 25% higher compared with that of leaves of small trees of F. sylvatica and Q. robur (2m height). The sucrose concentration in the cytosol of mesophyll cells from tall trees was between 120 and 240mM and about 4- to 8-fold lower than the sucrose concentration in the phloem sap of saplings. The analyzed SUT sequences of both tree species cluster into three types, similar to SUTs from other plant species. Heterologous expression in yeast confirmed that all analyzed SUTs are functional sucrose transporters. Moreover, all SUTs were expressed in leaves, bark and wood of the canopy and the expression levels in small and tall trees were similar. The results show that the phloem loading in leaves of tall trees of F. sylvatica and Q. robur probably involves active steps, because there is an uphill concentration gradient for sucrose. SUTs may be involved in phloem loading.

Sugar profiles modulation of mangoes during osmotic dehydration in agave syrup solutions.

Chemical interaction and multicompound competition were investigated on solids gain and carbohydrate profiles evolution during osmotic dehydration of mangoes. Tommy Atkins mango slices (0.4cm and 1.5cm thickness) were osmotically processed at 40°C for up to 4h and 8h, respectively. Osmotic solutions (60 °Brix) were separated in two categories: single solute (sucrose, glucose, fructose) and multisolute (agave syrup, alone or with additions of 5% inulin or 0.1-0.3% xanthan gum) solutions. High performance liquid chromatography (HPLC) analysis was carried out on treated mango to determine sugar profiles evolution during osmotic dehydration and final product concentrations. Findings pointed out that composition of osmotic solution may modulate mango sugar profiles by triggering uptake or loss of sugar according to different phenomena: chemical potential gradient, lixiviation, prevailing mass transfer, formation of carbohydrate barrier, and solution viscosity. Mango was enriched with the solute present in the single solute osmotic solution, while it lost its own native sugars, which were absent in the osmotic solution. Increasing sample thickness reduces individual sugar uptake or loss in mango treated with both single and multisolute solutions. Significant differences in mono solute solution behavior were found for sucrose due to its capability to form a sugar layer outside the surface of thicker samples, which was shown by scanning electron microscopy (SEM) images, a barrier markedly hindering the sucrose uptake or loss. Addition of polysaccharides (particularly xanthan gum) was found to have an impact of lowering mango individual sugar uptake (18-30%). Practical Application These results will help in understanding the mechanisms by which gain of individual sugars could be reduced and composition could be modulated during osmotic dehydration of fruits. Thus, the findings in this work could lead to production of low-sugar content, osmotically processed mango snacks, enriched with inulin, enhancing their dietary and marketable value.

OsDOF11 Promotes Crown Root Formation via Cytokinin in Oryza Sativa.

Crown root is the main part of root system, which performs an important role in rice growth and development, especially in nutrition and water assimilation. Previously, we reported negative feedback regulation loop between Oryza sativa DNA BINDING WITH ONE FINGER 11 (OsDOF11) and cytokinin by Oryza sativa CYTOKININ OXIDASE/DEHYDROGENASE 4 (OsCKX4) in rice development. Reverse transcription quantitative RT-PCR analyses was used to analyze the related gene transcript level. Nitrogen and hormone were measured by CHN-Nitrogen analyser and Liquid chromatography mass spectrometer, respectively. Exogenous application of cytokinin and [13C] sucrose labeled stable isotope uptake experiments help us to explain the relationship between OsDOF11 and cytokinin. We demonstrate the role of OsDOF11 in root development. We note that the loss function of OsDOF11 displays the reduced crown roots number, low activity of nitrogen assimilation and low content of cytokinin and auxin. The expression level of WUSCHEL-related homeobox (OsWOX11), A-type response regulator 2 (OsRR2), OsRR3, and OsCKX4 were decreased in osdof11-1, as well as in OsDOF11 RNA interference 9 mutants (RNAi-9 lines). Through Exogenous application of multiple concentrations of cytokinin as treatment to osdof11-1 mutant, RNAi-9 lines, and wild type (WT). We found that the crown roots number of osdof11-1 plants were rescued as the cytokinin concentration increased gradually from 1 μM to 10 μM, but the effect was weaker in RNAi-9 line. And cytokinin inhibited sucrose uptake activity from Murashige-Skoog medium with 3.0% sucrose (MS30) by OsDOF11 in rice root. OsDOF11 promotes crown root formation via cytokinin in oryza sativa. These results provide a physiological basis for further analysis of the OsDOF11 function of in rice root development.

Inhibition of HSP90 Preserves Blood-Brain Barrier Integrity after Cortical Spreading Depression.

Cortical spreading depression (CSD) is a pathophysiological mechanism underlying headache disorders, including migraine. Blood–brain barrier (BBB) permeability is increased during CSD. Recent papers have suggested that heat shock proteins (HSP) contribute to the integrity of the blood–brain barrier. In this study, the possible role of HSP90 in CSD-associated blood–brain barrier leak at the endothelial cell was investigated using an in vitro model, for the blood–endothelial barrier (BEB), and an in vivo model with an intact BBB. We measured barrier integrity using trans endothelial electric resistance (TEER) across a monolayer of rodent brain endothelial cells (bEnd.3), a sucrose uptake assay, and in situ brain perfusion using female Sprague Dawley rats. CSD was induced by application of 60 mM KCl for 5 min in in vitro experiments or cortical injection of KCl (1 M, 0.5 µL) through a dural cannula in vivo. HSP90 was selectively blocked by 17-AAG. Our data showed that preincubation with 17-AAG (1 µM) prevented the reduction of TEER values caused by the KCl pulse on the monolayer of bEnd.3 cells. The elevated uptake of 14C-sucrose across the same endothelial monolayer induced by the KCl pulse was significantly reduced after preincubation with HSP90 inhibitor. Pre-exposure to 17-AAG significantly mitigated the transient BBB leak after CSD induced by cortical KCl injection as determined by in situ brain perfusion in female rats. Our results demonstrated that inhibition of HSP90 with the selective agent 17-AAG reduced CSD-associated BEB/BBB paracellular leak. Overall, this novel observation supports HSP90 inhibition mitigates KCl-induced BBB permeability and suggests the development of new therapeutic approaches targeting HSP90 in headache disorders.

Probing Genome-Scale Model Reveals Metabolic Capability and Essential Nutrients for Growth of Probiotic Limosilactobacillus reuteri KUB-AC5.

Simple SummaryConsiderable attention has been given to the species Limosilactobacillus reuteri regarding its probiotic potential. Here, the genome-scale metabolic model (GSMM) of L. reuteri KUB-AC5, namely iTN656 was developed and exploited to evaluate the metabolic capability of L. reuteri KUB-AC5 under various carbon sources. The simulated growth behaviors of iTN656 under single and double omission analysis promisingly identified 14 essential single nutrients and 2 essential double nutrients (pairwise glutamine-glutamate and asparagine-aspartate) for L. reuteri KUB-AC5 growth. Moreover, the integrated transcriptomic analysis using iTN656 allowed to probe the potential metabolic routes for enhancing growth of L. reuteri KUB-AC5 involved in sucrose uptake, nucleotide biosynthesis, urea cycle, and glutamine transporter. Overall, iTN656 provides a powerful systems biology platform for gaining insights into cell metabolism of lactic acid bacteria and guiding the rational development of synthetic media design for scale-up of probiotic production in industrial scale.Limosilactobacillus reuteri KUB-AC5 displays the hallmark features of probiotic properties for food and feed industries. Optimization of cultivation condition for the industrial production is important to reach cell concentration and cost reduction. Considering the strain-specific growth physiology, metabolic capability, and essential nutrients of L. reuteri KUB-AC5, the genome-scale metabolic model (GSMM) of L. reuteri KUB-AC5 was developed. Hereby, the GSMM of iTN656 was successfully constructed which contained 656 genes, 831 metabolites, and 953 metabolic reactions. The iTN656 model could show a metabolic capability under various carbon sources and guide potentially 14 essential single nutrients (e.g., vitamin B complex and amino acids) and 2 essential double nutrients (pairwise glutamine-glutamate and asparagine-aspartate) for L. reuteri KUB-AC5 growth through single and double omission analysis. Promisingly, the iTN656 model was further integrated with transcriptome data suggesting that putative metabolic routes as preferable paths e.g., sucrose uptake, nucleotide biosynthesis, urea cycle, and glutamine transporter for L. reuteri KUB-AC5 growth. The developed GSMM offers a powerful tool for multi-level omics analysis, enabling probiotic strain optimization for biomass overproduction on an industrial scale.

Loss of Sucrase-Isomaltase Function Increases Acetate Levels and Improves Metabolic Health in Greenlandic Cohorts

The sucrase-isomaltase (SI) c.273_274delAG loss-of-function variant is common in Arctic populations and causes congenital sucrase-isomaltase deficiency, which is an inability to break down and absorb sucrose and isomaltose. Children with this condition experience gastrointestinal symptoms when dietary sucrose is introduced. We aimed to describe the health of adults with sucrase-isomaltase deficiency. The association between c.273_274delAG and phenotypes related to metabolic health was assessed in 2 cohorts of Greenlandic adults (n= 4922 and n= 1629). A sucrase-isomaltase knockout (Sis-KO) mouse model was used to further elucidate the findings. Homozygous carriers of the variant had a markedly healthier metabolic profile than the remaining population, including lower body mass index (β [standard error], -2.0 [0.5] kg/m2; P= 3.1× 10-5), body weight (-4.8 [1.4] kg; P= 5.1× 10-4), fat percentage (-3.3% [1.0%]; P= 3.7× 10-4), fasting triglyceride (-0.27 [0.07] mmol/L; P= 2.3× 10-6), and remnant cholesterol (-0.11 [0.03] mmol/L; P= 4.2× 10-5). Further analyses suggested that this was likely mediated partly by higher circulating levels of acetate observed in homozygous carriers (β [standard error], 0.056 [0.002] mmol/L; P= 2.1× 10-26), and partly by reduced sucrose uptake, but not lower caloric intake. These findings were verified in Sis-KO mice, which, compared with wild-type mice, were leaner on a sucrose-containing diet, despite similar caloric intake, had significantly higher plasma acetate levels in response to a sucrose gavage, and had lower plasma glucose level in response to a sucrose-tolerance test. These results suggest that sucrase-isomaltase constitutes a promising drug target for improvement of metabolic health, and that the health benefits are mediated by reduced dietary sucrose uptake and possibly also by higher levels of circulating acetate.

The Acquisition of the scr Gene Cluster Encoding Sucrose Metabolization Enzymes Enables Strains of Vibrio parahaemolyticus and Vibrio vulnificus to Utilize Sucrose as Carbon Source.

Most strains of Vibrio parahaemolyticus are unable to utilize sucrose as carbon source, though few exceptions exist. We investigated a sucrose-positive V. parahaemolyticus strain by whole-genome sequencing (WGS) and confirmed the presences of a genomic island containing sucrose utilization genes. A 4.7 kb DNA cluster consisting of three genes: scrA encoding a sucrose uptake protein, scrK encoding a fructokinase, and scrB coding for a sucrose-6-phosphate hydrolase, was PCR amplified and inserted into the Vibrio/Escherichia coli shuttle vector pVv3. Two recombinant plasmids, only differing in the orientation of the insert with respect to the pVv3-lacZα-fragment, conferred the E. coli K12 transformants the ability to utilize sucrose. The introduction of the two plasmids into sucrose-negative V. parahaemolyticus and V. vulnificus strains also results in a change of the sucrose utilization phenotype from negative to positive. By performing a multiplex PCR targeting scrA, scrK, and scrB, 43 scr-positive V. parahaemolyticus isolates from our collection of retail strains were detected and confirmed to be able to use sucrose as carbon source. Strains unable to utilize the disaccharide were negative by PCR for the scr genes. For in-depth characterization, 17 sucrose-positive V. parahaemolyticus were subjected to WGS. A genomic island with a nucleotide identity of >95% containing scrA, scrB, scrK and three additional coding sequences (CDS) were identified in all strains. The additional genes were predicted as a gene coding for a transcriptional regulator (scrR), a porin encoding gene and a CDS of unknown function. Sequence comparison indicated that the genomic island was located in the same region of the chromosome II in all analyzed V. parahaemolyticus strains. Structural comparison of the genomes with sequences of the sucrose utilizing species V. alginolyticus revealed the same genomic island, which indicates a possible distribution of this genetic structure by horizontal gene transfer. The comparison of all genome sequences based on SNP differences reveals that the presence of sucrose utilizing genes is found in genetically diverse V. parahaemolyticus strains and is not restricted to a subset of closely related strains.