Sorbitol Destroyed Intestinal Microfold Cells (M Cells) Development through Inhibition of PDE4-Mediated RANKL Expression.

Objective: Microfold cells (M cells) are specific intestinal epithelial cells for monitoring and transcytosis of antigens, microorganisms, and pathogens in the intestine. However, the mechanism for M-cell development remained elusive. Materials and Methods: Real-time polymerase chain reaction, immunofluorescence, and western blotting were performed to analyze the effect of sorbitol-regulated M-cell differentiation in vivo and in vitro, and luciferase and chromatin Immunoprecipitation were used to reveal the mechanism through which sorbitol-modulated M-cell differentiation. Results: Herein, in comparison to the mannitol group (control group), we found that intestinal M-cell development was inhibited in response to sorbitol treatment as evidenced by impaired enteroids accompanying with decreased early differentiation marker Annexin 5, Marcksl1, Spib, sox8, and mature M-cell marker glycoprotein 2 expression, which was attributed to downregulation of receptor activator of nuclear factor kappa-В ligand (RANKL) expression in vivo and in vitro. Mechanically, in the M-cell model, sorbitol stimulation caused a significant upregulation of phosphodiesterase 4 (PDE4) phosphorylation, leading to decreased protein kinase A (PKA)/cAMP-response element binding protein (CREB) activation, which further resulted in CREB retention in cytosolic and attenuated CREB binds to RANKL promoter to inhibit RANKL expression. Interestingly, endogenous PKA interacted with CREB, and this interaction was destroyed by sorbitol stimulation. Most importantly, inhibition of PDE4 by dipyridamole could rescue the inhibitory effect of sorbitol on intestinal enteroids and M-cell differentiation and mature in vivo and in vitro. Conclusion: These findings suggested that sorbitol suppressed intestinal enteroids and M-cell differentiation and matured through PDE4-mediated RANKL expression; targeting to inhibit PDE4 was sufficient to induce M-cell development.

Maltose accumulation-induced cell death in Saccharomyces cerevisiae.

Pretreatment of lignocellulose yields a complex sugar mixture that potentially can be converted into bioethanol and other chemicals by engineered yeast. One approach to overcome competition between sugars for uptake and metabolism is the use of a consortium of specialist strains capable of efficient conversion of single sugars. Here, we show that maltose inhibits cell growth of a xylose-fermenting specialist strain IMX730.1 that is unable to utilize glucose because of the deletion of all hexokinase genes. The growth inhibition cannot be attributed to a competition between maltose and xylose for uptake. The inhibition is enhanced in a strain lacking maltase enzymes (dMalX2) and completely eliminated when all maltose transporters are deleted. High-level accumulation of maltose in the dMalX2 strain is accompanied by a hypotonic-like transcriptional response, while cells are rescued from maltose-induced cell death by the inclusion of an extracellular osmolyte such as sorbitol. These data suggest that maltose-induced cell death is due to high levels of maltose uptake causing hypotonic-like stress conditions and can be prevented through engineering of the maltose transporters. Transporter engineering should be included in the development of stable microbial consortia for the efficient conversion of lignocellulosic feedstocks.

Dipeptidyl peptidase-4 inhibitor and sodium-glucose cotransporter 2 inhibitor additively ameliorate hepatic steatosis through different mechanisms of action in high-fat diet-fed mice.

AIM: Dipeptidyl peptidase-4 (DPP-4) inhibitors suppress the inactivation of incretin hormones and lower blood glucose levels by inhibiting DPP-4 function. Sodium-glucose cotransporter 2 (SGLT2) inhibitors lower blood glucose levels in an insulin-independent manner by inhibiting renal reabsorption of glucose. DPP-4 and SGLT2 inhibitors each have the potential to improve hepatic steatosis; however, their combined effects remain unclear. In this study, we examined the effects of the combination of these drugs on hepatic steatosis using high-fat diet-fed mice. METHOD: C57BL/6J male mice were fed a 60% high-fat diet for 2 months to induce hepatic steatosis. Mice were divided into four groups (control; DPP-4 inhibitor anagliptin; SGLT2 inhibitor luseogliflozin; anagliptin and luseogliflozin combination), and the effects of each drug and their combination on hepatic steatosis after a 4-week intervention were evaluated. RESULTS: There were no differences in blood glucose levels among the four groups. Anagliptin suppresses inflammation- and chemokine-related gene expression. It also improved macrophage fractionation in the liver. Luseogliflozin reduced body weight, hepatic gluconeogenesis and blood glucose levels in the oral glucose tolerance test. The combination treatment improved hepatic steatosis without interfering with the effects of anagliptin and luseogliflozin, respectively, and fat content and inflammatory gene expression in the liver were significantly improved in the combination group compared with the other groups. CONCLUSION: The combination therapy with the DPP-4 inhibitor anagliptin and the SGLT2 inhibitor luseogliflozin inhibits fat deposition in the liver via anti-inflammatory effects during the early phase of diet-induced liver steatosis.

Long-term sorbitol consumption affects the hippocampus and alters cognitive function in aged mice.

The systemic effects of the artificial sweetener sorbitol on older adult individuals have not been elucidated. We assessed the effects of sorbitol consumption on cognitive and gingival health in a mouse model. Aged mice were fed 5% sorbitol for 3 months before their behavior was assessed, and brain and gingival tissues were collected. Long-term sorbitol consumption inhibited gingival tissue aging in aged mice. However, it caused cognitive decline and decreased brain-derived neurotrophic factor (BDNF) in the hippocampus. Sorbitol consumption did not affect homeostatic function; however, it may exert effects within the brain, particularly in the hippocampus.

Widely distributable and retainable in-situ gelling material for treating myocardial infarction.

Intramyocardial hydrogel injection is a promising therapy to prevent negative remodeling following myocardial infarction (MI). In this study, we report a mechanism for in-situ gel formation without external stimulation, resulting in an injectable and tissue-retainable hydrogel for MI treatment, and investigate its therapeutic outcomes. A liquid-like polymeric solution comprising poly(3-acrylamidophenylboronic acid-co-acrylamide) (BAAm), polyvinyl alcohol (PVA), and sorbitol (S) increases the viscous modulus by reducing the pre-added sorbitol concentration is developed. This solution achieves a sol-gel transition in-vitro in heart tissue by spontaneously diffusing the sorbitol. After intramyocardial injection, the BAAm/PVA/S with lower initial viscous modulus widely spreads in the myocardium and gelate compared to a viscoelastic alginate (ALG) hydrogel and is retained longer than the BAAm/S solution. Serial echocardiogram analyses prove that injecting the BAAm/PVA/S into the hearts of subacute MI rats significantly increases the fraction shortening and ejection shortening and attenuates the expansion of systolic LV diameter for up to 21 d after injection compared to the saline injection as a control, but the ALG injection does not. In addition, histological evaluation shows that only the BAAm/PVA/S decreases the infarct size and increases the wall thickness 21 d after injection. The BAAm/PVA/S intramyocardial injection is better at restraining systolic ventricular dilatation and cardiac failure in the rat MI model than in the control groups. Our findings highlight an effective injectable hydrogel therapy for MI by optimizing injectability-dependent distribution and retention of injected material. STATEMENT OF SIGNIFICANCE: In-situ gelling material is a promising strategy for intramyocardial hydrogel injection therapy for myocardial infarction (MI). Since the sol-gel transition of reported materials is driven by external stimulation such as temperature, pH, or ultraviolet, their application in vivo remains challenging. In this study, we first reported a synthetic in-situ gelling material (BAAm/PVA/S) whose gelation is stimulated by spontaneously reducing pre-added sorbitol after contacting the heart tissue. The BAAm/PVA/S solution spreads evenly, and is retained for at least 21 d in the heart tissue. Our study demonstrated that intramyocardial injection of the BAAm/PVA/S with more extensive distribution and longer retention had better effects on preventing LV dilation and improving cardiac function after MI than that of viscoelastic ALG and saline solution. We expect that these findings provide fundamental information for the optimum design of injectable biomaterials for treating MI.

Gelatin and gelatin/starch-based films modified with sorbitol for wound healing.

Gelatin-based films modified with sorbitol were produced from gelatin solution or gelatin/starch blends using a simple and low-cost solvent casting method, and subsequently, their physicochemical, mechanical, and biocompatibility properties were characterized. This work focused on developing and optimizing a biopolymeric blend to improve the pure biopolymers' properties for potential biomedical applications such as wound dressing. The films were characterized in terms of morphology and transparency, mechanical, moisture and swelling properties, thermal stability, and degradation potential. Moreover, hemocompatibility, as well as cytocompatibility of prepared films, were examined. The addition of sorbitol contributed to improving mechanical properties, swelling reduction, and increasing biostability over time. The cytocompatibility of obtained films was confirmed in vitro with two different human cell lines, fibroblastic and osteoblastic, and a more favorable cellular response was received for fibroblasts. Further, in hemocompatibility studies, it was found that all films may be classified as non-hemolytic as they did not have a negative effect on the human erythrocytes. The obtained results indicate the great potential of the gelatin/starch blends modified with sorbitol as regenerative biomaterials intended for wound healing applications.

Identification of Potential Aldose Reductase Inhibitors Using Convolutional Neural Network-Based in Silico Screening.

Aldose reductase (ALR2) is a notable enzyme of the polyol pathway responsible for aggravating diabetic neuropathy complications. The first step begins when it catalyzes the reduction of glucose to sorbitol with NADPH as a coenzyme. Elevated concentrations of sorbitol damage the tissues, leading to complications like neuropathy. Though considerable effort has been pushed toward the successful discovery of potent inhibitors, its discovery still remains an elusive task. To this end, we present a 3D convolutional neural network (3D-CNN) based ALR2 inhibitor classification technique by dealing with snapshots of images captured from 3D chemical structures with multiple rotations as input data. The CNN-based architecture was trained on the 360 sets of image data along each axis and further prediction on the Maybridge library by each of the models. Subjecting the retrieved hits to molecular docking leads to the identification of the top 10 molecules with high binding affinity. The hits displayed a better blood-brain barrier penetration (BBB) score (90% with more than four scores) as compared to standard inhibitors (38%), reflecting the superior BBB penetrating efficiency of the hits. Followed by molecular docking, the biological evaluation spotlighted five compounds as promising ALR2 inhibitors and can be considered as a likely prospect for further structural optimization with medicinal chemistry efforts to improve their inhibition efficacy and consolidate them as new ALR2 antagonists in the future. In addition, the study also demonstrated the usefulness of scaffold analysis of the molecules as a method for investigating the significance of structurally diverse compounds in data-driven studies. For reproducibility and accessibility purposes, all of the source codes used in our study are publicly available.

1,5-Anhydro-d-glucitol derivative and galloylated flavonoids isolated from the leaves of Acer ginnala Maxim. as dual inhibitors of PTP1B and α-glucosidase enzymes: In vitro and in silico studies.

Four undescribed compounds (two 1,5-anhydro-d-glucitol derivatives and two galloyl derivatives) and fourteen known compounds were isolated and structurally identified from leaves of Acer ginnala Maxim. (Amur maple). Structures and absolute configurations of the four undescribed compounds were determined using extensive analysis of NMR spectroscopic, HRESI-MS, modified Mosher ester method, and comparison with spectroscopic data of known compounds. Bioactivity evaluation revealed that the isolated 1,5-anhydro-d-glucitol derivative, galloylated flavonol rhamnosides, and galloylated flavanols had inhibitory effects on both protein tyrosine phosphatase-1B (PTP1B, IC50 values ranging of 3.46-12.65 µM) and α-glucosidase (IC50 values ranging of 0.88-6.06 µM) in comparison with a positive control for PTP1B (ursolic acid, IC50 = 5.10 µM) or α-glucosidase (acarbose, IC50 = 141.62 µM). A combination of enzyme kinetic analysis and molecular docking provided additional evidence in favor of their inhibitory activities and mechanism. These data demonstrate that A. ginnala Maxim. together with its constituents are promising sources of potent candidates for developing novel anti-diabetic medications.

SnRK1 kinase-mediated phosphorylation of transcription factor bZIP39 regulates sorbitol metabolism in apple.

Sorbitol is a major photosynthate produced in leaves and transported through the phloem of apple (Malus domestica) and other tree fruits in Rosaceae. Sorbitol stimulates its own metabolism, but the underlying molecular mechanism remains unknown. Here, we show that sucrose nonfermenting 1 (SNF1)-related protein kinase 1 (SnRK1) is involved in regulating the sorbitol-responsive expression of both SORBITOL DEHYDROGENASE 1 (SDH1) and ALDOSE-6-PHOSPHATE REDUCTASE (A6PR), encoding 2 key enzymes in sorbitol metabolism. SnRK1 expression is increased by feeding of exogenous sorbitol but decreased by sucrose. SnRK1 interacts with and phosphorylates the basic leucine zipper (bZIP) transcription factor bZIP39. bZIP39 binds to the promoters of both SDH1 and A6PR and activates their expression. Overexpression of SnRK1 in 'Royal Gala' apple increases its protein level and activity, upregulating transcript levels of both SDH1 and A6PR without altering the expression of bZIP39. Of all the sugars tested, sorbitol is the only 1 that stimulates SDH1 and A6PR expression, and this stimulation is blocked by RNA interference (RNAi)-induced repression of either SnRK1 or bZIP39. These findings reveal that sorbitol acts as a signal regulating its own metabolism via SnRK1-mediated phosphorylation of bZIP39, which integrates sorbitol signaling into the SnRK1-mediated sugar signaling network to modulate plant carbohydrate metabolism.

Effects of Magnesium on nitrate uptake and sorbitol synthesis and translocation in apple seedlings.

Both magnesium (Mg) and nitrogen (N) play many important roles in plant physiological and biochemical processes. Plants usually exhibit low nitrogen utilization efficiency (NUE) under Mg deficiency conditions, but the mechanisms by which Mg regulates NUE are not well understood. Herein, we investigated biomass, nutrient uptake, sorbitol and sucrose transport, and relative gene expression in apple seedlings under various concentrations of Mg and N treatments in hydroponic cultures. We first observed that low Mg supply significantly limited plant growth and N, Mg concentrations. Increasing the supply of N, but not Mg, partially alleviated the inhibition of plant growth under low Mg stress, which indicated that Mg deficiency had a negative impact on plant growth because it inhibits N absorption. Moreover, we found that the expression of nitrate transporter genes MdNRT2.1 and MdNRT2.4 was significantly downregulated by low Mg stress, and sufficient Mg significantly promoted sucrose and sorbitol synthesis and transport from leaves to roots by regulating relevant enzyme activity and genes expression. Further experiments showed that exogenous sorbitol could rapidly restore MdNRT2.1/2.4 expression and nitrate uptake under low Mg availability without increasing internal Mg level, suggesting that Mg may regulate MdNRT2.1/2.4 expression by regulating more sorbitol transport to roots, the effect of Mg on N was indirect, sorbitol played a key role during this process. Taken together, Mg promoted sorbitol synthesis and transport into roots, thus upregulating the expression of MdNRT2.1/2.4 and increasing the absorption of nitrate.

Cryo-derived plants through embryogenesis showed same levels of vinblastine and vincristine (anticancer) in Catharanthus roseus and had normal genome size.

Cryopreservation of rare plant materials is an important approach for preserving germplasms and is a good added concept to tissue banking. The preservation of embryogenic cell suspensions is even more valuable as the tissues facilitate in producing millions of embryos, plantlets and generates transgenics en masse. Catharanthus roseus is a medicinally important plant that produces a variety of anticancerous phytocompounds and needs conservation of alkaloid producing cell lines. In this study, embryogenic tissue banking has been attempted in C. roseus by the two-step cryopreservation method combining cryoprotection and dehydration. Prior to plunging into liquid nitrogen (LN), the tissues were exposed to osmotic-and cryoprotective agents. Two osmotic agents (sugar and sorbitol) and three cryoprotective compounds, polyethylene glycol (PEG), dimethyl sulfoxide (DMSO) and glycerol were used at varying concentrations to protect cells from freezing damages. Both sucrose and sorbitol increased callus biomass post-cryopreservation; the influence of sucrose was however, more prominent. Embryogenic tissue treated in medium with 0.4 M sucrose for 2 days followed by 5% PEG for 2 h showed maximum viability before (83%) and after (55%) cryopreservation, high regrowth percentage (77%) and produced an average 9 cell colonies per Petri dish. Additionally, dehydration (1-5 h) was tested to reduce water content for improving viability and regrowth of cryopreserved embryogenic cells. Among the various tested cryoprotective conditions, the highest (72%) viability was observed following the combination of treatments with 0.4 M sucrose (2 days),10% PEG (2 h) and dehydration (2 h). Maximum regrowth percentage (88%) and 12 colonies/petri dish was noted in combination of 0.4 M sucrose + 5% PEG. The cryopreserved calli differentiated into somatic embryos (52.78-54.33 globular embryos/callus mass) in NAA (0.5 mg/l) and BAP (0.5-1.0 mg/l) added media. Plantlets were successfully regenerated from cryopreserved tissue and the 2C DNA was estimated through flow cytometry. The genome size of cryopreserved regenerant was 1.51 pg/2C, which is similar to field-grown Catharanthus plants. Vinblastine and vincristine levels were nearly the same in mother plant's and frozen (cryopreserved) leaf tissue. The post cryopreservation embryogenesis protocol may be used for continuous production of plants for future applications.

Novel topical esmolol hydrochloride improves wound healing in diabetes by inhibiting aldose reductase, generation of advanced glycation end products, and facilitating the migration of fibroblasts.

Aims/Objectives: Wound healing in people with diabetes is delayed secondary to impaired nitric oxide generation, advanced glycation end products (AGE), and poor migration of epithelial cells. We developed a novel topical esmolol hydrochloride (Galnobax) and assessed its efficacy for wound healing in streptozocin-induced diabetic hairless rat. Methods: All experiments were performed at an animal laboratory and tertiary-care research facility. Ex vivo aldose reductase inhibition was assessed from enzymes obtained from a bacterial culture (spectrophotometer), sorbitol content in homogenized red blood cells, and AGE in glucose and bovine serum by fluorometry following the addition of esmolol in varying concentrations. A scratch assay of human fibroblasts, endothelial cells, and keratinocytes was assessed under a high-glucose environment and after esmolol by phase-contrast microscopy. The efficacy evaluation of the topical application of Galnobax (14 and 20%) or vehicle was conducted in streptozotocin-induced diabetic hairless rats, and endogenous nitrite and hydroxyproline from homogenized wound tissue were measured along with pharmacokinetic and dermal toxicity in Hanford miniature swine. Results: Esmolol inhibited the formation of sorbitol by 59% in erythrocytes in comparison to glucose-induced sorbitol levels. AGE generation in bovine serum albumin was reduced at 1 mM esmolol concentrations (2.6 ± 1.7) compared with control (p < 0.05) and similar to that of diclofenac (2.5 ± 1.3). Esmolol at 1 and 10 µM enhanced the migration of fibroblasts, epithelial cells, and keratinocytes compared with control. The nitric oxide levels (day 7) were 44 and 112% higher with Galnobax (14%) than those of the diabetic group (p < 0.05) and the vehicle control group (p < 0.05), respectively. The days 7 and 14 hydroxyproline in the wound was higher by 22 and 44% following Galnobax (14%) compared with the diabetic and vehicle control groups. The wound area exhibited better reduction with Galnobax at 14% up to day 10 follow-up compared with the controls. The pharmacokinetic and dermal toxicity in miniature swine suggested no significant adverse event with Galnobax. Conclusions: Topical esmolol hydrochloride is a novel, safe, and effective treatment modality that acts through pleotropic mechanisms to hasten wound healing in diabetes.

Prevention of hospital pathogen biofilm formation by antimicrobial peptide KWI18.

This study investigated the antimicrobial and antibiofilm activity of KWI18, a new synthetic peptide. KWI18 was tested against planktonic cells and Pseudomonas aeruginosa and Candida parapsilosis biofilms. Time-kill and synergism assays were performed. Sorbitol, ergosterol, lipid peroxidation, and protein oxidation assays were used to gain insight into the mechanism of action of the peptide. Toxicity was evaluated against erythrocytes and Galleria mellonella. KWI18 showed antimicrobial activity, with minimum inhibitory concentration (MIC) values ranging from 0.5 to 10 µM. KWI18 at 10 × MIC reduced P. aeruginosa and C. parapsilosis biofilm formation and cell viability. Time-kill assays revealed that KWI18 inhibited the growth of P. aeruginosa in 4 h and that of C. parapsilosis in 6 h. The mechanism of action was related to ergosterol as well as induction of oxidative damage in cells and biofilms. Furthermore, KWI18 demonstrated low toxicity to erythrocytes and G. mellonella. KWI18 proved to be an effective antibiofilm agent, opening opportunities for the development of new antimicrobials.

Isoflavaspidic Acid PB Extracted from Dryopteris fragrans (L.) Schott Inhibits Trichophyton rubrum Growth via Membrane Permeability Alternation and Ergosterol Biosynthesis Disruption.

Isoflavaspidic acid PB (PB), a phloroglucinol derivative extracted from aerial parts of Dryopteris fragrans (L.) Schott, had antifungal activity against several dermatophytes. This study was aimed at exploring the antifungal mechanism of PB against Trichophyton rubrum (T. rubrum). The effectiveness of PB in inhibiting T. rubrum growth was detected by time-kill kinetics study and fungal biomass determination. Studies on the mechanism of action were investigated using scanning electron microscopy (SEM), transmission electron microscopy (TEM), sorbitol and ergosterol assay, nucleotide leakage measurement, and UPLC-based test and enzyme-linked immunosorbent assay. Fungicidal activity of PB was concentration- and time-dependent at 2 × MIC (MIC: 20 µg/mL) after 36 h. The total biomass of T. rubrum was reduced by 64.17%, 77.65%, and 84.71% in the presence of PB at 0.5 × MIC, 1 × MIC, and 2 × MIC, respectively. SEM analysis showed that PB changed mycelial morphology, such as shrinking, twisting, collapsing, and even flattening. TEM images of treated cells exhibited abnormal distributions of polysaccharide particles, plasmolysis, and cytoplasmic content degradation accompanied by plasmalemma disruption. There were no changes in the MIC of PB in the presence of sorbitol. However, the MIC values of PB were increased by 4-fold with exogenous ergosterol. At 4 h and 8 h, PB increased nucleotide leakage. Besides, ergosterol content in T. rubrum membrane treated with PB at 0.5 × MIC, 1 × MIC, and 2 × MIC was decreased by 9.58%, 15.31%, and 76.24%, respectively. There was a dose-dependent decrease in the squalene epoxidase (SE) activity. And the reduction in the sterol 14α-demethylase P450 (CYP51) activity was achieved after PB treatments at 1 × MIC and 2 × MIC. These results suggest that PB displays nonspecific action on the cell wall. The membrane damaging effects of PB were attributed to binding with ergosterol to increase membrane permeability and interfering ergosterol biosynthesis involved with the reduction of SE and CYP51 activities. Further study is needed to develop PB as a natural antifungal candidate for clinical use.

Long-term consumption of the sugar substitute sorbitol alters gut microbiome and induces glucose intolerance in mice.

AIMS: Epidemic obesity and diabetes have led to increased use of low-calorie sweeteners. Although several studies have suggested that consumption of artificial sweeteners, such as aspartame and saccharin, might have negative effects, the potential impacts of natural sweeteners on human health remain largely unknown. MAIN METHODS: The deferential effects of short term and long term consumption of sorbitol on glucose homeostasis in mice by oral gavage. The glucose homeostasis and utility were evaluated by both oral or intraperitoneal glucose tolerance tests. Insulin levels were determined using enzyme-linked immunosorbent assay. Changes of gut microbiome were evaluated by 16S rRNA gene sequencing, and analyzed by principal components analysis. KEY FINDINGS: Bolus feeding of sorbitol by gavage significantly increased plasma insulin concentrations and decreased fasting blood glucose levels. Intriguingly, long-term sorbitol gavage for four weeks showed no significant effects on intraperitoneal glucose tolerance test outcomes, but it induced glucose intolerance according to the oral glucose tolerance test. Thus, we tested whether long-term sorbitol gavage might alter the relative abundances of gut microbiome constituents in mice. Principal components analysis indicated that long-term sorbitol intake indeed caused significant changes to the gut microbiome. In particular, we found that long-term sorbitol intake significantly decreased the relative abundances of Bifidobacterium, Lachnospiraceae UCG 001, Lachnospiraceae NK4A136, Eubacterium ventriosum, Candidatus Arthromitus, and Ruminococcus torques. We also found that long-term sorbitol increased the relative abundances of Helicobacter, Tyzzerella, Alistipes, and Prevotella 9. SIGNIFICANCE: Long-term sorbitol consumption may change the composition of the gut microbiome and potentially induce glucose intolerance.

Licogliflozin for nonalcoholic steatohepatitis: a randomized, double-blind, placebo-controlled, phase 2a study.

Nonalcoholic steatohepatitis (NASH) is a common chronic liver disease that may advance to fibrosis and lead to mortality; however, no pharmacotherapy is currently available. We tested the hypothesis that inhibition of both the sodium-glucose cotransporters 1 and 2 with licogliflozin would lead to improvement in NASH. A total of 107 patients with phenotypic or histologic NASH were randomized (1:2:2) to receive oral administration of either placebo (n = 21), licogliflozin 30 mg (n = 43) or 150 mg (n = 43) once daily for 12 weeks. Licogliflozin 150 mg showed a significant 32% (80% confidence interval (CI): 21-43%; P = 0.002) placebo-adjusted reduction in serum alanine aminotransferase after 12 weeks of treatment, the primary endpoint of the study. However, the 30 mg dose of licogliflozin did not meet the primary endpoint (placebo-adjusted reduction 21% (80% CI: 7-32%; P = 0.061)). Diarrhea occurred in 77% (33 of 43), 49% (21 of 43) and 43% (9 of 21) of patients treated with licogliflozin 150 mg, 30 mg and placebo, respectively, which was mostly mild in severity. No other major safety concerns were identified. Treatment with 150 mg licogliflozin led to reductions in serum alanine aminotransferase in patients with NASH. Studies of longer duration and in combination with drugs that have different mechanisms of action are needed to validate these findings and to define a role of licogliflozin as a therapeutic option for NASH. ClinicalTrials.gov identifier: NCT03205150.

Using Live Imaging and Fluorescence Ubiquitinated Cell Cycle Indicator Embryonic Stem Cells to Distinguish G1 Cell Cycle Delays for General Stressors like Perfluoro-Octanoic Acid and Hyperosmotic Sorbitol or G2 Cell Cycle Delay for Mutagenic Stressors like Benzo(a)pyrene.

Lowest observable adverse effects level (LOAEL) is a standard point-of-departure dose in toxicology. However, first observable adverse effects level (FOAEL) was recently reported and is used, in this study, as one criterion to detect a mutagenic stimulus in a live imager. Fluorescence ubiquitinated cell cycle indicator (FUCCI) embryonic stem cells (ESC) are green in the S-G2-M phase of the cell cycle and not green in G1-phase. Standard media change here is a mild stress that delays G1-phase and media change increases green 2.5- to 5-fold. Since stress is mild, media change rapidly increases green cell number, but higher stresses of environmental toxicants and positive control hyperosmotic stress suppress increased green after media change. Perfluoro-octanoic acid (PFOA) and diethyl phthalate (DEP) previously suppressed progression of nongreen to green cell cycle progression. Here, bisphenol A (BPA), cortisol, and positive control hyperosmotic sorbitol also suppress green fluorescence, but benzo(a)pyrene (BaP) at high doses (10 µM) increases green fluorescence throughout the 74-h exposure. Since any stress can affect many cell cycle phases, messenger RNA (mRNA) markers are best interpreted in ratios as dose-dependent mutagens increase in G2/G1 and nonmutagens increase G1/G2. After 74-h exposure, RNAseq detects G1 and G2 markers and increasing BaP doses increase G2/G1 ratios but increasing hyperosmotic sorbitol and PFOA doses increase G1/G2 marker ratios. BaP causes rapid green increase in FOAEL at 2 h of stimulus, whereas retinoic acid caused significant green fluorescence increases only late in culture. Using a live imager to establish FOAEL and G2 delay with FUCCI ESC is a new method to allow commercial and basic developmental biologists to detect drugs and environmental stimuli that are mutagenic. Furthermore, it can be used to test compounds that prevent mutations. In longitudinal studies, uniquely provided by this viable reporter and live imager protocol, follow-up can be done to test whether the preventative compound itself causes harm.

Lipofundin mediates major inhibition of intravenous propofol on phorbol myristate acetate and Escherichia coli-induced neutrophil extracellular traps.

BACKGROUND: Neutrophil extracellular traps (NETs) consist of chromatin DNA networks that are studded with cytosolic and granular antimicrobial proteins to trap or kill an infected microorganism. A lipid emulsion, the solvent of pure propofol for intravenous application, is given to clinical patients who require intravenous feeding of fatty acids and fat for energy. Intravenous propofol is widely used to sedate critically ill patients. Both intravenous propofol and its lipid emulsion have immunomodulatory activity. However, the role of lipid emulsion of intravenous propofol on NET induction remains unclear. METHODS: In this study, neutrophils were stimulated with phorbol myristate acetate (PMA) or Escherichia coli (E. coli) in the absence or presence of intravenous propofol (Propofol-Lipuro®), its solvent lipid emulsion (Lipofundin) or pure propofol, and NETs were stained with SYTOX Green for visualization and quantification. Total HOCl was determined by measuring the taurine-chloramine complex, and intracellular HOCl was evaluated with BioTracker™ TP-HOCl 1 dye. RESULTS: PMA-induced NETs were not efficiently inhibited when Propofol-Lipuro® was added after PMA stimulation. Clinically relevant concentrations of Lipofundin exerted a significant reduction in PMA-induced NETs and total reactive oxidative species (ROS), which was comparable to that observed for Propofol-Lipuro®. Lipofundin transiently reduced intracellular HOCl production and the phosphorylation level of extracellular regulated kinase (p-ERK) but did not scavenge HOCl. Moreover, Lipofundin decreased E. coli-induced NETs in a ROS-independent pathway, similar to Propofol-Lipuro®. CONCLUSIONS: All data agree that Lipofundin, the major component of Propofol-Lipuro®, inhibits intracellular HOCl and p-ERK to suppress PMA-induced NET formation but reduces E.coli-induced NETs in a ROS-independent pathway.

Effect of Sorbitol on Alpha-Crystallin Structure and Function.

Loss of eye lens transparency due to cataract is the leading cause of blindness all over the world. While aggregation of lens crystallins is the most common endpoint in various types of cataracts, chaperone-like activity (CLA) of α-crystallin preventing protein aggregation is considered to be important for maintaining the eye lens transparency. Osmotic stress due to increased accumulation of sorbitol under hyperglycemic conditions is believed to be one of the mechanisms for diabetic cataract. In addition, compromised CLA of α-crystallin in diabetic cataract has been reported. However, the effect of sorbitol on the structure and function of α-crystallin has not been elucidated yet. Hence, in the present exploratory study, we described the effect of varying concentrations of sorbitol on the structure and function of α-crystallin. Alpha-crystallin purified from the rat lens was incubated with varying concentrations of sorbitol in the dark under sterile conditions for up to 5 days. At the end of incubation, structural properties and CLA were evaluated by spectroscopic methods. Interestingly, different concentrations of sorbitol showed contrasting results: at lower concentrations (5 and 50 mM) there was a decrease in CLA and subtle alterations in secondary and tertiary structure but not at higher concentrations (500 mM). Though, these results shed a light on the effect of sorbitol on α-crystallin structure-function, further studies are required to understand the mechanism of the observed effects and their implication to cataractogenesis.

Sorbitol Reduces Sensitivity to Alternaria by Promoting Ceramide Kinases (CERK) Expression through Transcription Factor Pswrky25 in Populus (Populus simonii Carr.).

Sugar, acting as a signal, can regulate the production of some chemical substance during plant defense responses. However, the molecular basis and regulatory mechanisms of sugar in poplar and other forest trees are still unclear. Sorbitol is a sugar-signaling molecule associated with plant defense. In this study, the pathogen-infested status of poplar was alleviated after exogenous feeding of 50 mM sorbitol. We sequenced and analyzed the transcriptome of poplar leaves before and after inoculation. The results showed that the genes PR1, WRKY, ceramide kinases (CERK) and so on responded to sorbitol feeding and pathogen infestation. We screened for genes related to disease resistance such as PsWRKY25 and PsCERK1 and found that significant disease spots occurred on day six of strep throat infestation. Under sorbitol feeding conditions, the appearance of spots was delayed after the pathogen inoculation. Due to the overexpression of PsWRKY25, the overexpression of PsCERK1 triggered the defense response in poplar. This was also confirmed by PsWRKY25 overexpression experiments. These findings present new insights into the influence of sorbitol on Populus simonii Carr. disease resistance. These results emphasize the value of molecular phenotypes in predicting physiological changes.