D-arabinose induces cell cycle arrest by promoting autophagy via p38 MAPK signaling pathway in breast cancer.

Breast cancer patients often have a poor prognosis largely due to lack of effective targeted therapy. It is now well established that monosaccharide enhances growth retardation and chemotherapy sensitivity in tumor cells. We investigated whether D-arabinose has capability to restrict the proliferation of tumor cells and its mechanism. Here, we report that D-arabinose induced cytotoxicity is modulated by autophagy and p38 MAPK signaling pathway in breast cancer cell lines. The proliferation of cells was evaluated by CCK-8 and Colony formation assay. The distribution of cells in cell cycle phases was analyzed by flow cytometry. Cell cycle, autophagy and MAPK signaling related proteins were detected by western blotting. Mouse xenograft model was used to evaluate the efficacy of D-arabinose in vivo. The proliferation of cells was dramatically inhibited by D-arabinose exposure in a dose-dependent manner, which was relevant to cell cycle arrest, as demonstrated by G2/M cell cycle restriction and ectopic expression of cell cycle related proteins. Mechanistically, we further identified that D-arabinose is positively associated with autophagy and the activation of the p38 MAPK signaling in breast cancer. In contrast, 3-Ma or SB203580, the inhibitor of autophagy or p38 MAPK, reversed the efficacy of D-arabinose. Additionally, D-arabinose in vivo treatment could significantly inhibit xenograft growth of breast cancer cells. Our findings were the first to reveal that D-arabinose triggered cell cycle arrest by inducing autophagy through the activation of p38 MAPK signaling pathway in breast cancer cells.

Effect of L-arabinose and lactulose combined with Lactobacillus plantarum on obesity induced by a high-fat diet in mice.

L-Arabinose, lactulose, and Lactobacillus plantarum (L. plantarum) have been reported to have glucolipid-lowering effects. Here, the effects of L-arabinose and lactulose combined with L. plantarum on obesity traits were investigated. According to the experimental results, the combination of L-arabinose, lactulose, and L. plantarum was more effective at reducing body weight, regulating glucolipid metabolism, and improving insulin resistance. Besides, this combination showed immunomodulatory activity by adjusting the T lymphocyte subsets and reduced the immune-related cytokine production. Moreover, it improved the gut barrier, ameliorated the disorder of gut microbiota, and upregulated the levels of SCFAs. More importantly, the AL group, LP group, and ALLP group showed different regulatory effects on the abundance of Bifidobacterium and Lactobacillus due to the presence of lactulose and L. plantarum. These findings elucidate that the combination of L-arabinose, lactulose, and L. plantarum constitutes a new synbiotic combination to control obesity by modulating glucolipid metabolism, immunomodulatory activity, inflammation, gut barrier, gut microbiota and production of SCFAs.

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.

Arabinose confers protection against intestinal injury by improving integrity of intestinal mucosal barrier.

There is a growing amount of research that highlights the significant involvement of metabolic imbalance and the inflammatory response in the advancement of colitis. Arabinose is a naturally occurring bioactive monosaccharide that plays a crucial role in the metabolic processes and synthesis of many compounds in living organisms. However, the more detailed molecular mechanism by which the administration of arabinose alleviates the progression of colitis and its associated carcinogenesis is still not fully understood. In the present study, arabinose is recognized as a significant and inherent protector of the intestinal mucosal barrier through its role in preserving the integrity of tight junctions within the intestines. Also, it is important to note that there is a positive correlation between the severity of inflammatory bowel disease (IBD) and colorectal cancer (CRC), as well as chemically-induced colitis in mice, and lower levels of arabinose in the bloodstream. In two mouse models of colitis, caused by dextran sodium sulfate (DSS) or by spontaneous colitis in IL-10-/- mice, damage to the intestinal mucosa was reduced by giving the mice arabinose. When arabinose is administrated to model with colitis, it sets off a chain of events that help keep the lysosomes together and stop cathepsin B from being released. During the progression of intestinal epithelial injury, this process blocks myosin light chain kinase (MLCK) from damaging tight junctions and causing mitochondrial dysfunction. In summary, the results of the study have provided evidence supporting the beneficial effects of arabinose in mitigating the progression of colitis. This is achieved through its ability to avoid dysregulation of the intestinal barrier. Consequently, arabinose may hold promise as a therapeutic supplementation for the management of colitis.

Arabinose- and xylose-modified analogs of 2',3'-cGAMP act as STING agonists.

Stimulator of interferon genes (STING) agonists are promising candidates for vaccine adjuvants and antitumor immune stimulants. The most potent natural agonist of STING, 2',3'-cyclic GMP-AMP (2',3'-cGAMP), is subject to nuclease-mediated inherent metabolic instability, thereby placing limits on its clinical efficacy. Here, we report on a new class of chemically synthesized sugar-modified analogs of 2',3'-cGAMP containing arabinose and xylose sugar derivatives that bind mouse and human STING alleles with high affinity. The co-crystal structures demonstrate that such analogs act as 2',3'-cGAMP mimetics that induce the "closed" conformation of human STING. These analogs show significant resistance to hydrolysis mediated by ENPP1 and increased stability in human serum, while retaining similar potency as 2',3'-cGAMP at inducing IFN-ß secretion from human THP1 cells. The arabinose- and xylose-modified 2',3'-cGAMP analogs open a new strategy for overcoming the inherent nuclease-mediated vulnerability of natural ribose cyclic nucleotides, with the additional benefit of high translational potential as cancer therapeutics and vaccine adjuvants.

Antiobesity effect of L-arabinose via ameliorating insulin resistance and modulating gut microbiota in obese mice.

OBJECTIVES: The global prevalence of obesity, a chronically trophic metabolic disease, has garnered significant attention. The aim of this study was to investigate L-arabinose, a unique functional sugar that improves insulin resistance and intestinal environment while promoting probiotic proliferation, for its potential in preventing obesity induced by a high-fat and high-sugar (HFHS) diet in mice. METHODS: The L-arabinose group was intragastrically administered with 0.4 mL 60 mg/(kg body weight) L-arabinose for 8 wk. The metformin group was intragastrically administered at 0.4 mL 300 mg/(kg body weight), as a positive control group. RESULTS: Treatment with L-arabinose resulted in a reduction of various obesity symptoms, such as prevented weight gain, increased liver-to-body ratio, decreased insulin, homeostasis model assessment for insulin resistance (HOMA-IR) index, and lipopolysaccharide (LPS) levels, as well as improved insulin resistance, reduced fat volume, inhibited hepatic steatosis, and repaired the pancreas. The L-arabinose treatment also improved lipid metabolism and inflammatory response, decreased the Firmicutes-to-Bacteroidetes ratio at the phylum level, and increased the relative abundance of Parabacteroides gordonii and Akkermansia muciniphila at the species level. CONCLUSION: Based on these results, L-arabinose could be a promising candidate for combating obesity and obesity-related diseases by regulating insulin resistance and gut microbiota.

Rare Sugar Metabolism and Impact on Insulin Sensitivity along the Gut-Liver-Muscle Axis In Vitro.

Rare sugars have recently attracted attention as potential sugar replacers. Understanding the biochemical and biological behavior of these sugars is of importance in (novel) food formulations and prevention of type 2 diabetes. In this study, we investigated whether rare sugars may positively affect intestinal and liver metabolism, as well as muscle insulin sensitivity, compared to conventional sugars. Rare disaccharide digestibility, hepatic metabolism of monosaccharides (respirometry) and the effects of sugars on skeletal muscle insulin sensitivity (impaired glucose uptake) were investigated in, respectively, Caco-2, HepG2 and L6 cells or a triple coculture model with these cells. Glucose and fructose, but not l-arabinose, acutely increased extracellular acidification rate (ECAR) responses in HepG2 cells and impaired glucose uptake in L6 cells following a 24 h exposure at 28 mM. Cellular bioenergetics and digestion experiments with Caco-2 cells indicate that especially trehalose (α1-1α), D-Glc-α1,2-D-Gal, D-Glc-α1,2-D-Rib and D-Glc-α1,3-L-Ara experience delayed digestion and reduced cellular impact compared to maltose (α1-4), without differences on insulin-stimulated glucose uptake in a short-term setup with a Caco-2/HepG2/L6 triple coculture. These results suggest a potential for l-arabinose and specific rare disaccharides to improve metabolic health; however, additional in vivo research with longer sugar exposures should confirm their beneficial impact on insulin sensitivity in humans.

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.

Oat ß-glucan and L-arabinose synergistically ameliorate glucose uptake in insulin-resistant HepG2 cells and exert anti-diabetic activity via activation of the PI3K/AKT pathway in db/db mice.

Oat ß-glucan (OBG) and L-arabinose (LA) have exhibited positive effects on diabetes and its complications. However, it is unclear whether OBG and LA have a synergistic effect. We investigated the effect of variable compositions (OBG : LA = 1 : 1, 1 : 2, 1 : 4,1 : 6, 1 : 8, 1 : 10, 2 : 1, 4 : 1, 6 : 1, 8 : 1, 10 : 1) on glucose uptake in IR-HepG2 cells induced by dexamethasone (DEX) to find out the optimal composition showing synergistic effects. Furthermore, this study evaluated the anti-diabetic activity of the optimal composition in db/db mice. In vitro, the OBG : LA = 1 : 1 group showed the strongest synergistic effects among the varied compositions, outperforming OBG and LA alone. In vivo, there were more beneficial effects in the OBG : LA = 1 : 1 group compared with the OBG and LA single-dosing groups. OBG : LA = 1 : 1 supplementation markedly decreased the levels of fasting blood glucose (FBG) and insulin (INS) in serum, improved glucose tolerance and insulin sensitivity, lowered blood lipid levels, and reduced liver lipid accumulation. Moreover, the western blot results indicated that the OBG : LA = 1 : 1 group up-regulated the protein expression of glucose transporter-4 (GLUT4), phosphatidylinositol 3-kinase (PI3K), and phospho-protein kinase B (p-AKT), while down-regulating the protein expression of phospho-phosphorylated insulin receptor substrate-1 (p-IRS1) to enhance insulin transduction in liver tissues. These findings suggest that OBG : LA = 1 : 1 synergistically ameliorated glucose metabolism disorders and alleviated insulin resistance by promoting the PI3K/AKT pathway in the liver.

Physicochemical properties and bioactivity of polysaccharides from Isaria cicadae Miquel with different extraction processes: effects on gut microbiota and immune response in mice.

The effect of different extraction processes on the physicochemical characterization, digestibility, antioxidant activity and prebiotic activity of Isaria cicadae Miquel (ICM) fruiting body polysaccharides was studied. Furthermore, the effect of ultrasound-assisted extraction of ICM (U-ICM) on gut microbiota, the intestinal barrier and immune response was deeply explored. This study found that ICMs showed high indigestibility in both α-amylase and artificial gastric juice, indicating that ICMs have the potential as dietary fiber. In contrast, U-ICM had the best antioxidant activity and prebiotic potential. Meanwhile, there was a structure-activity relationship between the antioxidant activity of ICMs and the content of uronic acid, arabinose and galactose. When healthy mice were fed U-ICM for 42 days, the relative abundances of Lactobacillus, Akkermansia, and Bacteroides were found to increase significantly, while that of Clostridium decreased significantly. Meanwhile, U-ICM significantly promotes the expression of tight junction protein and the production of cytokines, indicating that U-ICM had the function of enhancing the intestinal barrier and regulating the host immune response. In conclusion, U-ICM as dietary fiber has the potential to be developed as a gut health-promoting prebiotic component or functional food. This research provided a valuable resource for further exploring the structure-activity relationship and prebiotic activity of ICMs.

Chemical composition and adjuvant properties of the macromolecules from cultivated Cistanche deserticola Y. C. Ma as an immunopotentiator.

The study aims to investigate the constituents, adjuvant effects, and underlying mechanisms of purified polysaccharides from cultivated Cistanche deserticola (C. deserticola). Two macromolecules designated as CCDP-1 (26.5 kDa) and CCDP-2 (32.3 kDa) from C. deserticola were respectively identified as carbohydrate-lignin complexes with 44.1 % and 43.8 % lignin. CCDP-1 and CCDP-2 were composed of glucose, rhamnose, galactose, arabinose, and mannose respectively in the molar ratios of 7.22: 5.98:2.51:1.81:1.00 and 6.57:8.48:4.20:2.72:1.00. An in vitro experiment revealed that endotoxin-free CCDP-1 and CCDP-2 promoted splenocyte proliferation without cytotoxicity, but CCDP-2 induced dendritic cell (DC) maturation more efficiently than CCDP-1. An in vivo experiment suggested that CCDP-2 enhanced OVA-specific antibody production, antigen-specific T-cell activation, IFN-γ production, IL-4 production, and DC activation. Notably, CCDP-2 elicited a Th1-biased response. Mechanically, CCDP-2 upregulated CD40, CD80, CD86, and MHC II, facilitated allogeneic T-cell proliferation and Th1/Th2 cytokines, improved IFN-γ, IL-12, IL-6, and TNF-α production, and decreased endocytosis from DCs in vitro. Blocking assays indicated that TLR2 and TLR4 were the membrane receptor candidates of DCs. Western blot implied that CCDP-2 with the immune-enhancing activities were involved in the activation of MAPKs and NF-κB pathways in a dose-/time-related manner and could be employed as a more balanced Th1/Th2 adjuvant for vaccine exploitation.

Cooperative action of gut-microbiota-accessible carbohydrates improves host metabolic function.

Microbiota-accessible carbohydrates (MACs) exert health-promoting effects, but how each MAC impacts gut microbiota and regulates host physiology remains unclear. Here, we show that l-arabinose and sucrose cooperatively act on gut microbiota and exert anti-obesogenic effects. Specifically, l-arabinose, a monosaccharide that is poorly absorbed in the gut and inhibits intestinal sucrase, suppresses diet-induced obesity in mice in the presence of sucrose. Additionally, the suppressive effect of l-arabinose on adiposity is abrogated in mice lacking the short-chain fatty acid (SCFA) receptors GPR43 and GPR41. Mechanistically, l-arabinose increases the relative abundance of acetate and propionate producers (e.g., Bacteroides), while sucrose enhances SCFA production. Furthermore, l-arabinose and sucrose activate the glycolytic and pentose phosphate pathways of Bacteroides, respectively, indicating that they synergistically promote acetate production through distinct pathways. These findings suggest that each MAC has a unique property and thus may serve as a precision gut-microbiota modulator to promote host homeostasis.

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.

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.

Astrocyte-neuron lactate transport in the ACC contributes to the occurrence of long-lasting inflammatory pain in male mice.

Lactate transport is an important means of communication between astrocytes and neurons and is implicated in a variety of neurobiological processes. However, the connection between astrocyte-neuron lactate transport and nociceptive modulation has not been well established. Here, we found that Complete Freund's adjuvant (CFA)-induced inflammation pain leads to a significant increase in extracellular lactate levels in the anterior cingulate cortex (ACC). Inhibition of glycogenolysis and lactate release in the ACC disrupted the persistent, but not acute, inflammation pain induced by CFA, and this effect was reversed by exogenous L-lactate administration. Knocking down the expression of lactate transporters (MCT1, MCT4, or MCT2) also disrupted the long lasting inflammation pain induced by CFA. Moreover, glycogenolysis in the ACC is critical for the induction of molecular changes related to neuronal plasticity, including the induction of phospho- (p-) ERK, p-CREB, and Fos. Taken together, our findings indicate that astrocyte-neuron lactate transport in the ACC is critical for the occurrence of persistent inflammation pain, suggesting a novel mechanism underlying chronic pain.

A homogeneous polysaccharide from Lycium barbarum: Structural characterizations, anti-obesity effects and impacts on gut microbiota.

Lycium barbarum polysaccharides (LBPs) are known for their beneficial effects on diabetes, NAFLD and related chronic metabolic diseases induced by high-fat diet (HFD). However, the relevant researches are mainly about the whole crude polysaccharides, the specific active ingredient of LBPs and its bioactivity have been rarely explored. Herein, a homogeneous polysaccharide (LBP-W) was isolated and purified from crude LBPs. Structure characterizations indicated that LBP-W contained a main chain consisting of a repeated unit of →6)-ß-Galp(1 â†’ residues with branches composed of α-Araf, ß-Galp and α-Rhap residues at position C-3. The objective of this study was to evaluate the anti-obesogenic effect of LBP-W and figure out the underlying mechanisms. In vivo efficacy trial illustrated that LBP-W supplements can alleviate HFD-induced mice obesity significantly. Gut microbiota analysis showed that LBP-W not only improved community diversity of intestinal flora, but also regulated their specific genera. Moreover, LBP-W can increase the content of short-chain fatty acids (SCFAs), a metabolite of the intestinal flora. In summary, all these results demonstrated that the homogeneous polysaccharide purified from L. barbarum could be used as a prebiotic agent to improve obesity by modulating the composition of intestinal flora and the metabolism of SCFAs.

Synthesis and antitumor activity evaluation of oleanolic acid saponins bearing an acetylated l-arabinose moiety.

A series of oleanolic acid derivatives bearing acetyl-substituted l-arabinose moiety has been synthesized and screened in vitro for cytotoxicity against ten cancer cell lines and four normal cell lines. The antiproliferative evaluation indicated that synthetic derivatives showed excellent selectivity, as they were toxic against only A431 cell line. Among them, the compound 6 possesses the best inhibitory activity. A series of pharmacology experiments showed that compound 6 significantly induced A431 cells apoptosis and cell cycle arrest, which could serve as a promising lead candidate for further study.

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.

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.

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.