D-arabinose acts as antidepressant by activating the ACSS2-PPARγ/TFEB axis and CRTC1 transcription.

CREB-regulated transcription coactivator 1 (CRTC1), a pivotal synaptonuclear messenger, regulates synaptic plasticity and transmission to prevent depression. Despite exhaustive investigations into CRTC1 mRNA reductions in the depressed mice, the regulatory mechanisms governing its transcription remain elusive. Consequently, exploring rapid but non-toxic CRTC1 inducers at the transcriptional level is important for resisting depression. Here, we demonstrate the potential of D-arabinose, a unique monosaccharide prevalent in edible-medicinal plants, to rapidly enter the brain and induce CRTC1 expression, thereby eliciting rapid-acting and persistent antidepressant responses in chronic restrain stress (CRS)-induced depressed mice. Mechanistically, D-arabinose induces the expressions of peroxisome proliferator-activated receptor gamma (PPARγ) and transcription factor EB (TFEB), thereby activating CRTC1 transcription. Notably, we elucidate the pivotal role of the acetyl-CoA synthetase short-chain family member 2 (ACSS2) as an obligatory mediator for PPARγ and TFEB to potentiate CRTC1 transcription. Furthermore, D-arabinose augments ACSS2-dependent CRTC1 transcription by activating AMPK through lysosomal AXIN-LKB1 pathway. Correspondingly, the hippocampal down-regulations of ACSS2, PPARγ or TFEB alone failed to reverse CRTC1 reductions in CRS-exposure mice, ultimately abolishing the anti-depressant efficacy of D-arabinose. In summary, our study unveils a previously unexplored role of D-arabinose in activating the ACSS2-PPARγ/TFEB-CRTC1 axis, presenting it as a promising avenue for the prevention and treatment of depression.

Atomic force microscopy measurements and model of DNA bending caused by binding of AraC protein.

Atomic force microscopy (AFM) was used to conduct single-molecule imaging of protein/DNA complexes involved in the regulation of the arabinose operon of Escherichia coli. In the presence of arabinose, the transcription regulatory protein AraC binds to a 38 bp region consisting of the araI1 and araI2 half-sites. The domain positioning of full-length AraC, when bound to DNA, was not previously known. In this study, AraC was combined with 302 and 560 bp DNA and arabinose, deposited on a mica substrate, and imaged with AFM in air. High resolution images of 560 bp DNA, where bound protein was visible, showed that AraC induces a bend in the DNA with an angle 60° ± 12° with a median of 55°. These results are consistent with earlier gel electrophoresis measurements that measured the DNA bend angle based on migration rates. By using known domain structures of AraC, geometric constraints, and contacts determined from biochemical experiments, we developed a model of the tertiary and quaternary structure of DNA-bound AraC in the presence of arabinose. The DNA bend angle predicted by the model is in agreement with the measurement values. We discuss the results in view of other regulatory proteins that cause DNA bending and formation of the open complex to initiate transcription.

L-arabinose co-ingestion delays glucose absorption derived from sucrose in healthy men and women: a double-blind, randomised crossover trial.

Dietary interventions to delay carbohydrate digestion or absorption can effectively prevent hyperglycaemia in the early postprandial phase. L-arabinose can specifically inhibit sucrase. It remains to be assessed whether co-ingestion of L-arabinose with sucrose delays sucrose digestion, attenuates subsequent glucose absorption and impacts hepatic glucose output. In this double-blind, randomised crossover study, we assessed blood glucose kinetics following ingestion of a 200-ml drink containing 50 g of sucrose with 7·5 g of L-arabinose (L-ARA) or without L-arabinose (CONT) in twelve young, healthy participants (24 ± 1 years; BMI: 22·2 ± 0·5 kg/m2). Plasma glucose kinetics were determined by a dual stable isotope methodology involving ingestion of (U-13C6)-glucose-enriched sucrose, and continuous intravenous infusion of (6,6-2H2)-glucose. Peak glucose concentrations reached 8·18 ± 0·29 mmol/l for CONT 30 min after ingestion. In contrast, the postprandial rise in plasma glucose was attenuated for L-ARA, because peak glucose concentrations reached 6·62 ± 0·18 mmol/l only 60 min after ingestion. The rate of exogenous glucose appearance for L-ARA was 67 and 57 % lower compared with CONT at t = 15 min and 30 min, respectively, whereas it was 214 % higher at t = 150 min, indicating a more stable absorption of exogenous glucose for L-ARA compared with CONT. Total glucose disappearance during the first hour was lower for L-ARA compared with CONT (11 ± 1 v. 17 ± 1 g, P < 0·0001). Endogenous glucose production was not differentially affected at any time point (P = 0·27). Co-ingestion of L-arabinose with sucrose delays sucrose digestion, resulting in a slower absorption of sucrose-derived glucose without causing adverse effects in young, healthy adults.

Inhibitory effect of d-arabinose on oral bacteria biofilm formation on titanium discs.

OBJECTIVES: Biofilm formation on dental implant surfaces can cause peri-implant mucositis and peri-implantitis. Lectins are involved in interactions between bacteria or between bacteria and their hosts. Disrupting these interactions via specific sugars can result in reduced adhesion and biofilm formation. The purpose of this study was to identify sugars that function as antiadhesion or antibiofilm agents on titanium discs. METHODS: Of the sugars tested, the sugars that did not affect the planktonic growth of Streptococcus oralis, Fusobacterium nucleatum, and Porphyromonas gingivalis were selected. The selected sugars were assessed for their ability to inhibit biofilm formation of bacteria in single and consortium species by crystal violet staining, confocal laser scanning microscopy after live/dead staining, and scanning electron microscopy. The sugars were evaluated for their ability to inhibit activity of the quorum sensing molecule autoinducer 2 (AI-2) by bioluminescence assay. RESULTS: Biofilm formation of single bacteria or consortia of S. oralis, F. nucleatum, and P. gingivalis on titanium discs was significantly inhibited in the presence of d-arabinose. Pretreating titanium discs with d-arabinose for 3 min inhibited biofilm formation at a level comparable to that observed when d-arabinose was present over the entire period, suggesting that d-arabinose had initial anti-adhesive activity. In addition, d-arabinose inhibited the activity of AI-2. CONCLUSIONS: d-Arabinose may be a good candidate for application as an antibiofilm agent and AI-2 inhibitor.

Development of multiplexing gene silencing system using conditionally induced polycistronic synthetic antisense RNAs in Escherichia coli.

Although efficient methods of gene silencing have been established in eukaryotes, many different techniques are still used in bacteria due to the lack of a standardized tool. Here, we developed a convenient and efficient method to downregulate the expression of a specific gene using ∼140 nucleotide RNA with a 24-nucleotide antisense region from an arabinose-inducible expression plasmid by taking Escherichia coli lacZ and phoA genes encoding ß-galactosidase and alkaline phosphatase, respectively, as target genes to evaluate the model. We examined the antisense RNA (asRNA) design, including targeting position, uORF stability elements at the 5'-end, and Hfq-binding module at the 3'-end, and inducer amount required to obtain effective experimental conditions for gene silencing. Furthermore, we constructed multiplexed dual-acting asRNA genes in the plasmid, which were transcribed as polycistronic RNA and were able to knockdown multiple target genes simultaneously. We observed the highest inhibition level of 98.6% when lacZ was targeted using the pMKN104 asRNA expression plasmid, containing a five times stronger PBAD -10 promoter sequence with no requirement of the Hfq protein for repression. These features allow the system to be utilized as an asRNA expression platform in many bacteria, besides E. coli, for gene regulation.

Noradrenaline-induced l-lactate production requires d-glucose entry and transit through the glycogen shunt in single-cultured rat astrocytes.

During cognitive efforts mediated by local neuronal networks, approximately 20% of additional energy is required; this is mediated by chemical messengers such as noradrenaline (NA). NA targets astroglial aerobic glycolysis, the hallmark of which is the end product l-lactate, a fuel for neurons. Biochemical studies have revealed that astrocytes exhibit a prominent glycogen shunt, in which a portion of d-glucose molecules entering the cytoplasm is transiently incorporated into glycogen, a buffer and source of d-glucose during increased energy demand. Here, we studied single astrocytes by measuring cytosolic L-lactate ([lac]i ) with the FRET nanosensor Laconic. We examined whether NA-induced increase in [lac]i is influenced by: (a) 2-deoxy-d-glucose (2-DG, 3 mM), a molecule that enters the cytosol and inhibits the glycolytic pathway; (b) 1,4-dideoxy-1,4-imino-d-arabinitol (DAB, 300 µM), a potent inhibitor of glycogen phosphorylase and glycogen degradation; and (c) 3-nitropropionic acid (3-NPA, 1 mM), an inhibitor of the Krebs cycle. The results of these pharmacological experiments revealed that d-glucose uptake is essential for the NA-induced increase in [lac]i , and that this exclusively arises from glycogen degradation, indicating that most, if not all, d-glucose molecules in NA-stimulated cells transit the glycogen shunt during glycolysis. Moreover, under the defined transmembrane d-glucose gradient, the glycolytic intermediates were not only used to produce l-lactate, but also to significantly support oxidative phosphorylation, as demonstrated by an elevation in [lac]i when Krebs cycle was inhibited. We conclude that l-lactate production via aerobic glycolysis is an essential energy pathway in NA-stimulated astrocytes; however, oxidative metabolism is important at rest.

Tunable expression rate control of a growth-decoupled T7 expression system by L-arabinose only.

BACKGROUND: Precise regulation of gene expression is of utmost importance for the production of complex membrane proteins (MP), enzymes or other proteins toxic to the host cell. In this article we show that genes under control of a normally Isopropyl ß-D-1-thiogalactopyranoside (IPTG)-inducible PT7-lacO promoter can be induced solely with L-arabinose in a newly constructed Escherichia coli expression host BL21-AI, a strain based on the recently published approach of bacteriophage inspired growth-decoupled recombinant protein production. RESULTS: Here, we show that BL21-AI is able to precisely regulate protein production rates on a cellular level in an L-arabinose concentration-dependent manner and simultaneously allows for reallocation of metabolic resources due to L-arabinose induced growth decoupling by the phage derived inhibitor peptide Gp2. We have successfully characterized the system under relevant fed-batch like conditions in microscale cultivation (800 µL) and generated data proofing a relevant increase in specific yields for 6 different Escherichia coli derived MP-GFP fusion proteins by using online-GFP signals, FACS analysis, SDS-PAGE and western blotting. CONCLUSIONS: In all cases tested, BL21-AI outperformed the parental strain BL21-AI, operated in growth-associated production mode. Specific MP-GFP fusion proteins yields have been improved up to 2.7-fold. Therefore, this approach allows for fine tuning of MP production or expression of multi-enzyme pathways where e.g. particular stoichiometries have to be met to optimize product flux.

Fed-batch polyhydroxybutyrate production by Paraburkholderia sacchari from a ternary mixture of glucose, xylose and arabinose.

Polyhydroxybutyrate (PHB) is a biodegradable bioplastic that is comparable with many petroleum-based plastics in terms of mechanical properties and is highly biocompatible. Lignocellulosic biomass conversion into PHB can increase profit and add sustainability. Glucose, xylose and arabinose are the main monomer sugars derived from upstream lignocellulosic biomass processing. The sugar mixture ratios may vary greatly depending on the pretreatment and enzymatic hydrolysis conditions. Paraburkholderia sacchari DSM 17165 is a bacterium strain that can convert all three sugars into PHB. In this study, fed-batch mode was applied to produce PHB on three sugar mixtures (glucose:xylose:arabinose = 4:2:1, 2:2:1, 1:2:1). The highest PHB concentration produced was 67 g/L for 4:2:1 mixture at 41 h corresponding to an accumulation of 77% of cell dry weight as PHB. Corresponding sugar conversion efficiency and productivity were 0.33 g PHB/g sugar consumed and 1.6 g/L/h, respectively. The results provide references for process control to maximize PHB production from real sugar streams derived from corn fibre.

Adaptive and Mutational Responses to Peptide Dendrimer Antimicrobials in Pseudomonas aeruginosa.

Colistin (polymyxin E) is a last-resort antibiotic against multidrug-resistant isolates of Pseudomonas aeruginosa However, the nephro-toxicity of colistin limits its use, spurring the interest in novel antimicrobial peptides (AMP). Here, we show that the synthetic AMP-dendrimer G3KL (MW 4,531.38 Da, 15 positive charges, MIC = 8 mg/liter) showed faster killing than polymyxin B (Pmx-B) with no detectable resistance selection in P. aeruginosa strain PA14. Spontaneous mutants selected on Pmx-B, harboring loss of function mutations in the PhoQ sensor kinase gene, showed increased Pmx-B MICs and arnB operon expression (4-amino-l-arabinose addition to lipid A), but remained susceptible to dendrimers. Two mutants carrying a missense mutation in the periplasmic loop of the PmrB sensor kinase showed increased MICs for Pmx-B (8-fold) and G3KL (4-fold) but not for the dendrimer T7 (MW 4,885.64 Da, 16 positive charges, MIC = 8 mg/liter). The pmrB mutants showed increased expression of the arnB operon as well as of the speD2-speE2-PA4775 operon, located upstream of pmrAB, and involved in polyamine biosynthesis. Exogenous supplementation with the polyamines spermine and norspermine increased G3KL and T7 MICs in a phoQ mutant background but not in the PA14 wild type. This suggests that both addition of 4-amino-l-arabinose and secretion of polyamines are required to reduce susceptibility to dendrimers, probably neutralizing the negative charges present on the lipid A and the 2-keto-3-deoxyoctulosonic acid (KDO) sugars of the lipopolysaccharide (LPS), respectively. We further show by transcriptome analysis that the dendrimers G3KL and T7 induce adaptive responses through the CprRS two-component system in PA14.

High-level production of industrially relevant oxidases by a two-stage fed-batch approach: overcoming catabolite repression in arabinose-inducible Escherichia coli systems.

With the growing interest in enzyme applications, there is an urgent demand for economic, affordable, and flexible enzyme production processes. In the present paper, we developed a high cell density fed-batch process for the production of two cofactor-containing oxidase, 5-hydroxymethylfurfural oxidase (HMFO) and eugenol oxidase (EUGO). The approach involved the arabinose-inducible system to drive the expression while using mineral media. In order to overcome a major drawback of arabinose-inducible promoters, carbon catabolite repression, (CCR) by glucose, we developed a high cell density culture (HCDC), two-stage fed-batch protocol allowing us to reach cell densities exceeding 70 g/L of dry cell weight (DCW) using glucose as carbon source. Then, induction was achieved by adding arabinose, while changing the carbon source to glycerol. This strategy allowed us to obtain an eightfold increase in recombinant HMFO titer when compared with a reference batch fermentation in Erlenmeyer flasks using terrific broth (TB), typically used with arabinose-inducible strains. The optimized protocol was also tested for expression of a structurally unrelated oxidase, EUGO, where a similar yield was achieved. Clearly, this two-step protocol in which a relatively cheap medium (when compared to TB) can be used reduces costs and provides a way to obtain protein production levels similar to those of IPTG-based systems. KEY POINTS: • Arabinose promoters are not well suited for HCDC production due to CCR effect. • This drawback has been overcome by using a two-stage Fed-batch protocol. • Protein yield has been increased by an eightfold factor, improving process economics.

Inhibitory Activity of Quercetin 3-O-Arabinofuranoside and 2-Oxopomolic Acid Derived from Malus domestica on Soluble Epoxide Hydrolase.

Flavonoids and triterpenoids were revealed to be the potential inhibitors on soluble epoxide hydrolase (sEH). The aim of this study is to reveal sEH inhibitors from Fuji apples. A flavonoid and three triterpenoids derived from the fruit of Malus domestica were identified as quercetin-3-O-arabinoside (1), ursolic acid (2), corosolic acid (3), and 2-oxopomolic acid (4). They had half-maximal inhibitory concentration of the inhibitors (IC50) values of 39.3 ± 3.4, 84.5 ± 9.5, 51.3 ± 4.9, and 11.4 ± 2.7 µM, respectively, on sEH. The inhibitors bound to allosteric sites of enzymes in mixed (1) and noncompetitive modes (2-4). Molecular simulations were carried out for inhibitors 1 and 4 to calculate the binding force of ligands to receptors. The inhibitors bound to the left (1) and right (4) pockets next to the enzyme's active site. Based on analyses of their molecular docking and dynamics, it was shown that inhibitors 1 and 4 can stably bind sEH at 1 bar and 300 K. Finally, inhibitors 1 and 4 are promising candidates for further studies using cell-based assays and in vivo cardiovascular tests.

Characterizing the selectivity of ER α-glucosidase inhibitors.

The endoplasmic reticulum (ER) contains both α-glucosidases and α-mannosidases which process the N-linked oligosaccharides of newly synthesized glycoproteins and thereby facilitate polypeptide folding and glycoprotein quality control. By acting as structural mimetics, iminosugars can selectively inhibit these ER localized α-glycosidases, preventing N-glycan trimming and providing a molecular basis for their therapeutic applications. In this study, we investigate the effects of a panel of nine iminosugars on the actions of ER luminal α-glucosidase I and α-glucosidase II. Using ER microsomes to recapitulate authentic protein N-glycosylation and oligosaccharide processing, we identify five iminosugars that selectively inhibit N-glycan trimming. Comparison of their inhibitory activities in ER microsomes against their effects on purified ER α-glucosidase II, suggests that 3,7a-diepi-alexine acts as a selective inhibitor of ER α-glucosidase I. The other active iminosugars all inhibit α-glucosidase II and, having identified 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) as the most effective of these compounds, we use in silico modeling to understand the molecular basis for this enhanced activity. Taken together, our work identifies the C-3 substituted pyrrolizidines casuarine and 3,7a-diepi-alexine as promising "second-generation" iminosugar inhibitors.

Synthetic circuits reveal how mechanisms of gene regulatory networks constrain evolution.

Phenotypic variation is the raw material of adaptive Darwinian evolution. The phenotypic variation found in organismal development is biased towards certain phenotypes, but the molecular mechanisms behind such biases are still poorly understood. Gene regulatory networks have been proposed as one cause of constrained phenotypic variation. However, most pertinent evidence is theoretical rather than experimental. Here, we study evolutionary biases in two synthetic gene regulatory circuits expressed in Escherichia coli that produce a gene expression stripe-a pivotal pattern in embryonic development. The two parental circuits produce the same phenotype, but create it through different regulatory mechanisms. We show that mutations cause distinct novel phenotypes in the two networks and use a combination of experimental measurements, mathematical modelling and DNA sequencing to understand why mutations bring forth only some but not other novel gene expression phenotypes. Our results reveal that the regulatory mechanisms of networks restrict the possible phenotypic variation upon mutation. Consequently, seemingly equivalent networks can indeed be distinct in how they constrain the outcome of further evolution.

Arabinose biosynthesis is critical for salt stress tolerance in Arabidopsis.

The capability to maintain cell wall integrity is critical for plants to adapt to unfavourable conditions. l-Arabinose (Ara) is a constituent of several cell wall polysaccharides and many cell wall-localised glycoproteins, but so far the contribution of Ara metabolism to abiotic stress tolerance is still poorly understood. Here, we report that mutations in the MUR4 (also known as HSR8) gene, which is required for the biosynthesis of UDP-Arap in Arabidopsis, led to reduced root elongation under high concentrations of NaCl, KCl, NaNO3 , or KNO3 . The short root phenotype of the mur4/hsr8 mutants under high salinity is rescued by exogenous Ara or gum arabic, a commercial product of arabinogalactan proteins (AGPs) from Acacia senegal. Mutation of the MUR4 gene led to abnormal cell-cell adhesion under salt stress. MUR4 forms either a homodimer or heterodimers with its isoforms. Analysis of the higher order mutants of MUR4 with its three paralogues, MURL, DUR, MEE25, reveals that the paralogues of MUR4 also contribute to the biosynthesis of UDP-Ara and are critical for root elongation. Taken together, our work revealed the importance of the Ara metabolism in salt stress tolerance and also provides new insights into the enzymes involved in the UDP-Ara biosynthesis in plants.

Inadequate brain glycogen or sleep increases spreading depression susceptibility.

OBJECTIVE: Glycogen in astrocyte processes contributes to maintenance of low extracellular glutamate and K+ concentrations around excitatory synapses. Sleep deprivation (SD), a common migraine trigger, induces transcriptional changes in astrocytes, reducing glycogen breakdown. We hypothesize that when glycogen utilization cannot match synaptic energy demand, extracellular K+ can rise to levels that activate neuronal pannexin-1 channels and downstream inflammatory pathway, which might be one of the mechanisms initiating migraine headaches. METHODS: We suppressed glycogen breakdown by inhibiting glycogen phosphorylation with 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) and by SD. RESULTS: DAB caused neuronal pannexin-1 large pore opening and activation of the downstream inflammatory pathway as shown by procaspase-1 cleavage and HMGB1 release from neurons. Six-hour SD induced pannexin-1 mRNA. DAB and SD also lowered the cortical spreading depression (CSD) induction threshold, which was reversed by glucose or lactate supplement, suggesting that glycogen-derived energy substrates are needed to prevent CSD generation. Supporting this, knocking down the neuronal lactate transporter MCT2 with an antisense oligonucleotide or inhibiting glucose transport from vessels to astrocytes with intracerebroventricularly delivered phloretin reduced the CSD threshold. In vivo recordings with a K+ -sensitive/selective fluoroprobe, Asante Potassium Green-4, revealed that DAB treatment or SD caused a significant rise in extracellular K+ during whisker stimulation, illustrating the critical role of glycogen in extracellular K+ clearance. INTERPRETATION: Synaptic metabolic stress caused by insufficient glycogen-derived energy substrate supply can activate neuronal pannexin-1 channels as well as lower the CSD threshold. Therefore, conditions that limit energy supply to synapses (eg, SD) may predispose to migraine attacks, as suggested by genetic studies associating glucose or lactate transporter deficiency with migraine. Ann Neurol 2018;83:61-73.

Optimization of polyhydroxybutyrate production by experimental design of combined ternary mixture (glucose, xylose and arabinose) and process variables (sugar concentration, molar C:N ratio).

Conversion of lignocellulosic feedstocks to polyhydroxybutyrate (PHB) could make lignocellulosic biorefineries more profitable and sustainable. Glucose, xylose and arabinose are the main sugars derived from pretreatment and hydrolysis of herbaceous feedstocks. Burkholderia sacchari DSM 17165 is a bacterium that can convert these sugars into PHB. However, the effects of sugar ratio, sugar concentration, and molar C:N ratio on PHB production have not been studied. In this study, a seven-run mixture design for sugar ratio combined with a 32 full factorial design for process variables was performed to optimize PHB production. A polynomial model was built based on experimental data, and optimum conditions for different sugar streams were derived and validated. The highest PHB production (3.81 g/L) was achieved with arabinose at a concentration of 25.54 g/L and molar C:N ratio of 74.35. Results provide references for manipulation of sugar mixture and process control to maximize PHB production.

Enhanced production of recombinant Staphylococcus simulans lysostaphin using medium engineering.

Staphylococcus aureus, among other staphylococcal species, developed multidrug resistance and causes serious health risks that require complex treatments. Therefore, the development of novel and effective strategies to combat these bacteria has been gaining importance. Since Staphylococcus simulans lysostaphin is a peptidoglycan hydrolase effective against staphylococcal species, the enzyme has a significant potential for biotechnological applications. Despite promising results of lysostaphin as a bacteriocin capable of killing staphylococcal pathogens, it is still not widely used in healthcare settings due to its high production cost. In this study, medium engineering techniques were applied to improve the expression yield of recombinant lysostaphin in E. coli. A new effective inducible araBAD promoter system and different mediums were used to enhance lysostaphin production. Our results showed that the composition of autoinduction media enhanced the amount of lysostaphin production 5-fold with the highest level of active lysostaphin at 30 °C. The production cost of 1000 U of lysostaphin was determined as 4-fold lower than the previously proposed technologies. Therefore, the currently developed bench scale study has a great potential as a large-scale fermentation procedure to produce lysostaphin efficiently.

Oligodendrocyte Progenitor Cells Directly Utilize Lactate for Promoting Cell Cycling and Differentiation.

Oligodendrocyte progenitor cells (OPCs) undergo marked morphological changes to become mature oligodendrocytes, but the metabolic resources for this process have not been fully elucidated. Although lactate, a metabolic derivative of glycogen, has been reported to be consumed in oligodendrocytes as a metabolite, and to ameliorate hypomyelination induced by low glucose conditions, it is not clear about the direct contribution of lactate to cell cycling and differentiation of OPCs, and the source of lactate for remyelination. Therefore, we evaluated the effect of 1,4-dideoxy-1,4-imino-d-arabinitol (DAB), an inhibitor of the glycogen catabolic enzyme glycogen phosphorylase, in a mouse cuprizone model. Cuprizone induced demyelination in the corpus callosum and remyelination occurred after cuprizone treatment ceased. This remyelination was inhibited by the administration of DAB. To further examine whether lactate affects proliferation or differentiation of OPCs, we cultured mouse primary OPC-rich cells and analyzed the effect of lactate. Lactate rescued the slowed cell cycling induced by 0.4 mM glucose, as assessed by the BrdU-positive cell ratio. Lactate also promoted OPC differentiation detected by monitoring the mature oligodendrocyte marker myelin basic protein, in the presence of both 36.6 mM and 0.4 mM glucose. Furthermore, these lactate-mediated effects were suppressed by the reported monocarboxylate transporter inhibitor, α-cyano-4-hydroxy-cinnamate. These results suggest that lactate directly promotes the cell cycling rate and differentiation of OPCs, and that glycogen, one of the sources of lactate, contributes to remyelination in vivo. J. Cell. Physiol. 232: 986-995, 2017. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

Positron emission tomography probe demonstrates a striking concentration of ribose salvage in the liver.

PET is a powerful technique for quantifying and visualizing biochemical pathways in vivo. Here, we develop and validate a novel PET probe, [(18)F]-2-deoxy-2-fluoroarabinose ([(18)F]DFA), for in vivo imaging of ribose salvage. DFA mimics ribose in vivo and accumulates in cells following phosphorylation by ribokinase and further metabolism by transketolase. We use [(18)F]DFA to show that ribose preferentially accumulates in the liver, suggesting a striking tissue specificity for ribose metabolism. We demonstrate that solute carrier family 2, member 2 (also known as GLUT2), a glucose transporter expressed in the liver, is one ribose transporter, but we do not know if others exist. [(18)F]DFA accumulation is attenuated in several mouse models of metabolic syndrome, suggesting an association between ribose salvage and glucose and lipid metabolism. These results describe a tool for studying ribose salvage and suggest that plasma ribose is preferentially metabolized in the liver.

Breaking the Permeability Barrier of Escherichia coli by Controlled Hyperporination of the Outer Membrane.

In Gram-negative bacteria, a synergistic relationship between slow passive uptake of antibiotics across the outer membrane and active efflux transporters creates a permeability barrier, which efficiently reduces the effective concentrations of antibiotics in cells and, hence, their activities. To analyze the relative contributions of active efflux and the passive barrier to the activities of antibiotics, we constructed Escherichia coli strains with controllable permeability of the outer membrane. The strains expressed a large pore that does not discriminate between compounds on the basis of their hydrophilicity and sensitizes cells to a variety of antibacterial agents. We found that the efficacies of antibiotics in these strains were specifically affected by either active efflux or slow uptake, or both, and reflect differences in the properties of the outer membrane barrier, the repertoire of efflux pumps, and the inhibitory activities of antibiotics. Our results identify antibiotics which are the best candidates for the potentiation of activities through efflux inhibition and permeabilization of the outer membrane.