Chemical Composition, Antibacterial Properties, and Anti-Enzymatic Effects of Eucalyptus Essential Oils Sourced from Tunisia.

This study was conducted to examine the chemical composition of the essential oils (EOs) from six Tunisian Eucalyptus species and to evaluate their anti-enzymatic and antibiofilm activities. The EOs were obtained through hydro-distillation of dried leaves and subsequently analyzed using GC/MS. The main class of compounds was constituted by oxygenated monoterpenes, particularly prominent in E. brevifolia (75.7%), E. lehmannii (72.8%), and E. woollsiana (67%). Anti-enzymatic activities against cholinesterases, α-amylase, and α-glucosidase were evaluated using spectrophotometric methods. Notably, the E. brevifolia, E. extensa, E. leptophylla, E. patellaris, and E. woollsiana EOs displayed potent acetylcholinesterase (AChE) inhibition (IC50: 0.25-0.60 mg/mL), with E. lehmannii exhibiting lower activity (IC50: 1.2 mg/mL). E. leptophylla and E. brevifolia showed remarkable α-amylase inhibition (IC50: 0.88 mg/mL), while E. brevifolia and E. leptophylla significantly hindered α-glucosidase (IC50 < 30 mg/mL), distinguishing them from other EOs with limited effects. Additionally, the EOs were assessed for their anti-biofilm properties of Gram-positive (Staphylococcus aureus and Listeria monocytogenes) and Gram-negative (Acinetobacter baumannii, Pseudomonas aeruginosa and Escherichia coli) bacterial strains. The E. extensa EO demonstrated the main antibiofilm effect against E. coli and L. monocytogenes with an inhibition > 80% at 10 mg/mL. These findings could represent a basis for possible further use of Eucalyptus EOs in the treatment of human microbial infections and/or as a coadjutant in preventing and treating Alzheimer's disease and/or diabetes mellitus.

Digestive enzyme activity and macromolecule content in the hemolymph of differentially adapted Lymantria dispar L. populations after short-term increases in ambient temperature.

Global, unpredictable temperature increases have strong effects on all organisms, especially insects. Elucidating the effects of short-term temperature increases on midgut digestive enzymes (α-glucosidase, lipase, trypsin, and leucine aminopeptidase - LAP) and metabolic macromolecules in the hemolymph (proteins, lipids, and trehalose) of phytophagous pest larvae of Lymantria dispar is important for general considerations of insect adaptation to a warming climate and potential pest control options. We also wanted to determine whether the different adaptations of L. dispar populations to environmental pollution might affect their ability to cope with heat stress using larvae from the undisturbed, Kosmaj forest and disturbed, Lipovica forest. Heat treatments at 28 °C increased α-glucosidase activity in both larval populations, inhibited LAP activity in larvae from the polluted forest, and had no significant effect on trypsin and lipase activities, regardless of larval origin. The concentration of proteins, lipids, and trehalose in the hemolymph of larvae from the disturbed forest increased, whereas the population from the undisturbed forest showed only an increase in proteins and lipids after the heat treatments. Larval mass was also increased in larvae from the undisturbed forest. Our results suggest a higher sensitivity of digestive enzymes and metabolism to short-term heat stress in L. dispar populations adapted to pollution in their forest habitat, although climate warming is not beneficial even for populations from unpolluted forests. The digestive and metabolic processes of L. dispar larvae are substantially affected by sublethal short-term increases in ambient temperature.

In-depth in-vitro and in-vivo anti-diabetic evaluations of Fagonia cretica mediated biosynthesized selenium nanoparticles.

Therapeutic moieties derived from medicinal plants as well as plants-based ecofriendly processes for producing selenium nanoparticles have shown great promise in the management of type 2 diabetes mellitus (T2DM). The current study was aimed to assess the anti-diabetic potentials of Fagonia cretica mediated biogenic selenium nanoparticles (FcSeNPs) using in-vitro and in-vivo approaches. The bio-synthesized FcSeNPs were characterized using various techniques including UV-VIS spectrophotometry and FTIR analysis. The in-vitro efficacy of FcSeNPs were assessed against α-glucosidase, α-amylase enzymes as well as the anti-radical studies were performed using DPPH and ABTS free radicals scavenging assays. For in-vivo studies, 20 Male Balb/C albino-mice were randomly divided into 4 groups (n = 5) including normal group, disease group (Diabetic group with no treatment), control group and treatment group (Diabetic group treated with FcSeNPs). Further, biochemistry markers including pancreas, liver, kidney and lipid profile were assessed for all treatment groups. The FcSeNPs exhibited a dose-dependent inhibition against α-amylase and α-glucosidase at 62-1000 µg mL-1 concentration with IC50 values of 92 and 100 µg mL-1 respectively. In antioxidant experiments, the FcSeNPs demonstrated significant radicals scavenging effect against DPPH and ABTS radicals. In STZ-induced diabetic mice, a considerable decline in blood glucose level was observed after treatment with FcSeNPs. Anti-hyperglycemic effect of FcSeNPs treated animals were high (105 ± 3.22**) as compared to standard drug (128.6 ± 2.73** mg dL-1). Biochemical investigations revealed that all biochemical parameters for pancreas, liver function, renal function panel and lipid profile were significantly lowered in FcSeNPs treated animals. Our findings indicate a preliminary multi-target efficacy for FcSeNPs against type-2 diabetes and thus warrant further detailed studies.

Antidiabetic and anti-obesity properties of a polyphenol-rich flower extract from Tagetes erecta L. and its effects on Caenorhabditis elegans fat storages.

Diabetes mellitus (DM) is a metabolic disease characterized by a high blood sugar level that can cause severe complications to the organism or even death when not treated. However, certain dietary habits and foods may have beneficial effects on this condition. A polyphenolic-rich extract (containing hyperoside, isoquercitrin, quercetin, ellagic acid, and vanillic acid) of Tageres erecta L. (T. erecta) was obtained from yellow and orange flowers using an ethanolic Soxhlet extraction. These extracts were screened for antidiabetic and anti-obesity properties using in vitro and in vivo procedures. The capacity to inhibit the enzymes lipase and α-glucosidase, as well as the inhibition of advance glycation end-products (AGEs) was tested in vitro. Caenorhabditis elegans (C. elegans) was used as an obesity in vivo model to assess extracts effects on fat accumulation using the wild-type strain N2 and a mutant with no N3 fatty acid desaturase activity BX24. Extracts from both cultivars (yellow and orange) T. erecta presented in vitro inhibitory activity against the enzymes lipase and α-glucosidase, showing lower IC50 values than acarbose (control). They also showed important activity in preventing AGEs formation. The polyphenol-rich matrices reduced the fat content of obese worms in the wild-type strain (N2) down to levels of untreated C. elegans, with no significant differences found between negative control (100% reduction) and both tested samples (p < 0.05). Meanwhile, the fat reduction was considerably lower in the BX24 mutants (fat-1(wa-9)), suggesting that N3 fatty acid desaturase activity could be partially involved in the T. erecta flower effect. Our findings suggested that polyphenols from T. erecta can be considered candidate bioactive compounds in the prevention and improvement of metabolic chronic diseases such as obesity and diabetes.

Targeted delivery of Nitric Oxide triggered by α-Glucosidase to Ameliorate NSAIDs-induced Enteropathy.

Nonsteroidal anti-inflammatory drugs (NSAIDs) increase risks of severe small intestinal injuries. Development of effective therapeutic strategies to overcome this issue remains challenging. Nitric oxide (NO) as a gaseous mediator plays a protective role in small intestinal injuries. However, small intestine-specific delivery systems for NO have not been reported yet. In this study, we reported a small intestine-targeted polymeric NO donor (CS-NO) which was synthesized by covalent grafting of α-glucosidase-activated NO donor onto chitosan. In vitro and in vivo experiments demonstrated that CS-NO could be activated by intestinal α-glucosidase to release NO in the small intestine. Pre-treatment of mice with CS-NO significantly alleviated small intestinal damage induced by indomethacin, as demonstrated by down-regulation of the levels of pro-inflammatory cytokines and chemokines CXCL1/KC. Moreover, CS-NO also attenuated indomethacin-induced gut barrier dysfunction as evidenced by up-regulation of the levels of tight junction proteins and restoration of the levels of goblet cells and MUC2 production. Meanwhile, CS-NO effectively restored the defense function of Paneth cells against pathogens in small intestine. Our present study paves the way to develop NO-based therapeutic strategy for NSAIDs-induced small intestinal injuries.

Chemical Analysis and Investigation of Biological Effects of Salvia officinalis Essential Oils at Three Phenological Stages.

Salvia officinalis is a medicinal plant used to treat some diseases, including microbial infections and diabetes. Different studies showed the biological and pharmacological properties of this species. The aim of this study was the determination of the chemical compounds of S. officinalis essential oils and the investigation of their antimicrobial, antioxidant, antidiabetic, and anti-inflammatory properties. The chemical compounds of S. officinalis were determined by GC-MS analysis. The antioxidant activity was assessed by DPPH, ABTS, H2O2, and FRAP assays. The in vitro antidiabetic effect was evaluated by the inhibition of α-amylase, α-glucosidase, and lipase activities, and the anti-inflammatory effect was evaluated using the 5-lipoxygenase assay. Moreover, antibacterial activity was assessed against six bacterial strains using agar well diffusion assay and microdilution method. The main compounds in essential oils of S. officinalis at three phenological stages were naphthalenone, camphor, 1.8-cineole, and α-thujone. The full flowering stage essential oil showed the best antioxidant activity with different IC50 values according to the used tests. This oil also exhibited important inhibitory effects at the full flowering stage against α-amylase (IC50 = 69.23 ± 0.1 µg/mL), α-glucosidase (IC50 = 22.24 ± 0.07 µg/mL), and lipase (IC50 = 37.3 ± 0.03 µg/mL). The 5-lipoxygenase inhibitory effect was the best at the full flowering stage (IC50 = 9.24 ± 0.03 µg/mL). The results of the antibacterial evaluation revealed that, at three seasonal periods, S. officinalis essential oil demonstrated strong antibacterial activity. Although the full flowering stage had the best antibacterial activity, there were no significant differences between the three stages. Additionally, the essential oils showed bactericidal effects on Listeria monocytogenes, Staphylococcus aureus, Bacillus subtilis, Proteus mirabilis, Escherichia coli, and Salmonella typhimurium, respectively. The findings of this work showed remarkably that S. officinalis synthesizes essential oils according to different developmental stages. Moreover, it has exhibited interesting biological and pharmacological properties justifying its medicinal effects and suggesting it as a very important source of natural drugs.

Gut microbiota activity in chickens from two genetic lines and with outdoor-preferring, moderate-preferring, and indoor-preferring ranging profiles.

Despite the existing research into the gut microbiome of meat chickens, the associations between gut microbiome composition, its activity and chicken outdoor ranging frequency remain unexplored. The aim of this study was to determine the gut microbiota composition, activity and metabolic products in chickens of 2 different lines and 3 ranging profiles. Sixty non-beak trimmed birds, either Sasso or Green-legged Partridge were housed with access to outdoor ranges from wk. 5 to 10 of age. Outdoor ranges were video recorded to obtain frequencies of the birds' range use. The information about relative abundance of selected bacterial groups in the ceca including Lactobacillus spp., E. coli, Bifidobacterium spp., and Clostridium spp. was obtained with the PCR method. Gut microbiota activity was assessed based on the glycolytic activity of bacterial enzymes including, α-glucosidase, ß-glucosidase, α-galactosidase, ß-galactosidase, and ß-glucuronidase as well as based on the concentration of short-chain fatty acids (SCFA) in the caecal digesta. Statistical analysis was conducted by generalized linear mixed models, applying the breed and ranging profile as fixed effects and pen as a random factor. The lowest relative abundance of Bifidobacterium spp. was found in the cecal content of indoor-preferring Sasso birds (0.01 ± 0.001), as compared to all other birds in the experiment (ranging from 0.03 ± 0.01 to 0.11 ± 0.07; P = 0.0002). The lowest relative abundance of E. coli was identified for all outdoor-preferring birds and indoor- preferring Sasso birds (0.01 ± 0.001; P = 0.0087). Cecal activity of: α-glucosidase, ß-glucuronidase and ß-galactosidase was higher in Green-legged Partridges, as compared to Sasso (P = 0.013; P = 0.008; P = 0.004). Valeric acid concentrations were higher in moderate Green-legged Partridges than in Sasso of the same ranging profile (2.03 ± 0.16 vs. 1.5 ± 0.17; 0.016). The majority of the current results confirmed an effect of genotype and ranging profile on the various analyzed parameters. In outdoor-preferring birds, the consumption of pasture originating feed sources as a supplement to the indoor accessible cereal-based diet likely caused the positive effects on the birds' microbial profile.

Polycyclic Phenol Derivatives from the Leaves of Spermacoce latifolia and Their Antibacterial and α-Glucosidase Inhibitory Activity.

Three new polycyclic phenol derivatives, 2-acetyl-4-hydroxy-6H-furo [2,3-g]chromen-6-one (1), 2-(1',2'-dihydroxypropan-2'-yl)-4-hydroxy-6H-furo [2,3-g][1]benzopyran-6-one (2) and 3,8,10-trihydroxy-4,9-dimethoxy-6H-benzo[c]chromen-6-one (8), along with seven known ones (3-7, 9 and 10) were isolated for the first time from the leaves of Spermacoce latifolia. Their structures were determined by spectroscopic analysis and comparison with literature-reported data. These compounds were tested for their in vitro antibacterial activity against four Gram-(+) bacteria: Staphyloccocus aureus (SA), methicillin-resistant Staphylococcus aureus (MRSA), Bacillus cereus (BC), Bacillus subtilis (BS), and the Gram-(-) bacterium Escherichia coli. Compounds 1, 2, 5 and 8 showed antibacterial activity toward SA, BC and BS with MIC values ranging from 7.8 to 62.5 µg/mL, but they were inactive to MRSA. Compound 4 not only showed the best antibacterial activity against SA, BC and BS, but it further displayed significant antibacterial activity against MRSA (MIC 1.95 µg/mL) even stronger than vancomycin (MIC 3.9 µg/mL). No compounds showed inhibitory activity toward E. coli. Further bioassay indicated that compounds 1, 4, 5, 6, 8 and 9 showed in vitro α-glucosidase inhibitory activity, among which compound 9 displayed the best α-glucosidase inhibitory activity with IC50 value (0.026 mM) about 15-fold stronger than the reference compound acarbose (IC50 0.408 mM). These results suggested that compounds 4, 8 and 9 were potentially highly valuable compounds worthy of consideration to be further developed as an effective anti-MRSA agent or effective α-glucosidase inhibitors, respectively. In addition, the obtained data also supported that S. latifolia was rich in structurally diverse bioactive compounds worthy of further investigation, at least in searching for potential antibiotics and α-glucosidase inhibitors.

Screening of antimicrobial, antioxidant, antidiabetic activities, anatomical and morphological properties of Colchicum speciosum Steven (Colchicaceae).

Colchicum speciosum Steven species is a perennial stemless plant. C. speciosum is a flowering herb native to mountainous regions of northern Turkey, the Caucasus, and northern Iran. It has been known as "Vargit, Aci Çigdem, Güz Çigdemi". The present study reports the antimicrobial, antioxidant, α-amylase and α-glucosidase inhibitory activities of corm, leaf and flower methanol extracts, anatomical (light and electron microscopes) properties of root, corm, leaf, flowers and morphological characteristics of C. speciosum. Three different part of extracts C. speciosum were evaluated against E. coli ATCC 8739, S. aureus ATCC 6538, B. subtilis ATCC 19,659, C. albicans ATCC 10,231, C. krusei ATCC 14,243, and C. tropicalis ATCC 750. The most effective extract was found to be MeOH extract for corm and leaf against C. tropicalis ATCC 750 strain with MIC value 160 > µg/mL. It has been investigated first time anatomy of the tepal, ovary, anther, filament of C. speciosum. Leaf extract was the highest phenolic component (78.61842 µg GAE/ mg extract). As a result of DPPH• and ABTS•+ tests, it was determined that the leaf extract showed the best activity (IC50 = 6.568 µg/mL and IC50 = 3.243 µg/mL, respectively). Corm extract exhibited α-glucosidase inhibitory activity with an IC50 value of 21039 µg/mL. This is the first study of the in vitro antimicrobial, antioxidant, antidiabetic activities, detailed anatomical and morphological properties of C. speciosum. HiGHLiGHTS : • Antioxidant-antidiabetic-antimicrobial potential of Colchicum speciosum • Leaf extract had the highest phenolic component • The leaf got the highest DPPH• and ABTS•+ antioxidant potential • Corm extract exhibited α-glucosidase inhibitory activity • The most effective extract was found to be MeOH extract for corm and leaf against C. tropicalis • This is the first study of the in vitro antimicrobial, antioxidant, antidiabetic activities, detailed anatomical and morphological properties of C. speciosum.

Effect of environmental temperature on semen quality and seminal plasma metabolites of Mediterranean buffalo bulls.

High-quality semen with high viability is critical to improving the in-vitro fertilization efficiency. This study aimed to understand the effect of ambient temperature and humidity on semen quality and seminal plasma biochemical parameters of Mediterranean buffalo in March and July. The metabolites of seminal plasma in two seasons were detected using the UPLC-MS/MS method. The results showed that temperature and humidity index (THI) in March were 66.86 ± 2.98, and 82.94 ± 3.52 in July. Compared with in March, breath frequency, rectal temperature, and heat shock protein 70 expressions of seminal plasma were significantly increased in July (p < 0.05), motility of sperm was dramatically reduced, and sperm deformity rate was significantly increased (p < 0.05). Fructose, acid phosphatase and α-glucosidase in seminal plasma were significantly increased (p < 0.05) in July, while testosterone level was significantly reduced (p < 0.05). Six different metabolites were found in the two groups, which involved in three metabolic pathways, the tricarboxylic acid cycle, glycerophospholipid, glyoxylic acid and dicarboxylic acid. The above results indicate that the increased ambient temperature has obvious side effects on the semen quality of Mediterranean buffalo, and the compromised quality is associated with the change of metabolites related to male hormone secretion, energy metabolism and fatty acid oxidation.

Inhibitory activity of flavonoids against human sucrase-isomaltase (α-glucosidase) activity in a Caco-2/TC7 cellular model.

Type 2 diabetes (T2D) is the most common form of diabetes, and the number of people with this metabolic disease is steadily increasing worldwide. Among the available antidiabetic agents, α-glucosidase inhibitors are the most effective at reducing postprandial hyperglycaemia (PPHG), one of the main characteristics of T2D. However, most of the studies that have been performed have used the more readily available rat intestinal preparations or yeast α-glucosidase as the enzyme source, which despite being useful and cost effective, have a questionable physiological value. The present study evaluates the inhibitory activity of a selected group of flavonoids against human sucrase-isomaltase (SI), the α-glucosidase found in Caco-2/TC7 cells. A microassay using the physiological substrates sucrose and maltose, and a synthetic substrate, p-nitrophenyl-α-D-glucopyranoside (pNPG) was performed. The most active flavonoid was compound 4 (melanoxetin), presenting an IC50 value similar using the two natural substrates. In contrast, the tested flavonoids were not effective at inhibiting SI, when pNPG was used as a substrate. Hydroxylation of flavonoids at C-3 of the C ring, at C-3' and C-4' of the B ring, and at C-7 and C-8 of the A ring were the features that improved the inhibitory activity of flavonoids against human SI. These phenolic compounds deserve further exploration as alternatives to the currently available α-glucosidase inhibitors. The present study also demonstrates that the non-clinical in vitro studies conducted for the evaluation of α-glucosidase activity should use the human source rather than surrogate sources of α-glucosidase.

Avalglucosidase alfa: First Approval.

Avalglucosidase alfa (NEXVIAZYME™; avalglucosidase alfa-ngpt) is a hydrolytic lysosomal glycogen-specific recombinant human α-glucosidase product being developed by Sanofi Genzyme (formerly Genzyme Corporation) for the treatment of Pompe disease. Pompe disease is an autosomal recessive lysosomal storage disease caused by a deficiency of the lysosomal enzyme acid α-glucosidase (GAA), which results in intralysosomal accumulation of glycogen in various tissues. In August 2021, avalglucosidase alfa received its first approval in the USA for the treatment of patients 1 year of age and older with late-onset Pompe disease (GAA deficiency). In July 2021, avalglucosidase alfa received a positive opinion in the EU for long-term enzyme replacement therapy for the treatment of patients with Pompe disease. The drug is under regulatory review in the UK and Japan, and clinical studies are underway in several countries worldwide. This article summarizes the milestones in the development of avalglucosidase alfa leading to this first approval for late-onset Pompe disease.

Thermal treatment enhances the α-glucosidase inhibitory activity of bitter melon (Momordica charantia) by increasing the free form of phenolic compounds and the contents of Maillard reaction products.

Inhibition of α-glucosidase can slow carbohydrate metabolism, which is known as an effective strategy for diabetes treatment. The aim of this study is to evaluate the effect of thermal treatment (50, 60, and 70℃) for 15 days on the α-glucosidase inhibitory activity of bitter melon. The results show that the bitter melon heated at 70℃ for 12 days had the best α-glucosidase inhibitory effect. However, the amount of free polyphenols, 5-hydroxymethyl-2-furfural (5-HMF), and the browning degree of bitter melon generally increased with the time (15 days) and temperature of the thermal treatment, which is positively related to their antioxidant and α-glucosidase inhibitory activities. In conclusion, aged bitter melon shows great α-glucosidase inhibitory activity, which may be related to the increased free form of the involved phenolic compounds and Maillard reaction products. This suggests that thermal processing may be a good way to enhance the application of bitter melon for diabetes treatment. PRACTICAL APPLICATION: The thermal processing of bitter melon provides an application for diabetes treatment. This study demonstrated that heat-treated bitter melon can lower the blood glucose level; therefore, it can be used as a potential anti-hyperglycemic and functional food.

Bioactive stigmastadienone from Isodon rugosus as potential anticholinesterase, α-glucosidase and COX/LOX inhibitor: In-vitro and molecular docking studies.

Natural product is a well-known source of bioactive compounds. Herein, a steroidal compound stigmasta-7,22-diene-3-one (stigmastadienone) has been isolated from Isodon rugosus. The potency of isolated compound has been tested for several in-vitro targets. The acetyl and butyrylcholinesterase assays were performed using Ellman's procedure. For the in-vitro antidiabetic potential, α-glucosidase inhibitory assay was performed. Similarly, the cyclo and lipoxygenase pathways were studied to find its potential role in the management of inflammation and analgesia. The 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and hydrogen peroxide (H2O2) assays were performed for the antioxidant potentials. Docking studies were performed against acetylcholinesterase, cyclooxygenase and lipoxygenase targets. In anticholinesterase assays, stigmastadienone exhibited half-maximal inhibitory concentration (IC50) values of 13.52 and 11.53 µg/ml for acetyl and butyrylcholinesterase respectively. The observed IC50 values for that of galantamine were 6.07 and 4.42 µg/ml for acety and butyrylcholinesterase respectively. In inhibiting α-glucosidase enzyme, the compound showed mediocre IC50 of 109.40 µg/ml compared to the standard acarbose (7.60 µg/ml). The stigmastadienone proved to be an excellent inhibitor of cyclooxygenase 2 (COX-2) and 5-lipoxygenase (5-LOX) attaining IC50 values of 4.72 and 3.36 µg/ml respectively. The standard drugs IC50 values for COX-2 (celecoxib) and 5-LOX (montelukast) were 3.81 and 2.74 µg/ml respectively. The enzymatic activities of stigmastadienone were also supplemented with antioxidant results, specifically it was more dominant against DPPH and ABTS free radicals. Docking studies showed that only the carbonyl oxygen is able to form hydrogen bond interaction with the residues. In conclusions, the stigmastadienone has been isolated from Isodon rugosus for the first time. Moreover, the compound has been evaluated for several biochemical pathways which suggest its pharmacological role on the explored targets.

Iminosugar Glucosidase Inhibitors Reduce Hepatic Inflammation in Hepatitis A Virus-Infected Ifnar1-/- Mice.

Iminosugar compounds are monosaccharide mimetics with broad but generally weak antiviral activities related to inhibition of enzymes involved in glycobiology. Miglustat (N-butyl-1-deoxynojirimycin), which is approved for the treatment of lipid storage diseases in humans, and UV-4 [N-(9-methoxynonyl)-1-deoxynojirimycin] inhibit the replication of hepatitis A virus (HAV) in cell culture (50% inhibitory concentrations [IC50s] of 32.13 µM and 8.05 µM, respectively) by blocking the synthesis of gangliosides essential for HAV cell entry. We used a murine model of hepatitis A and targeted mass spectrometry to assess the capacity of these compounds to deplete hepatic gangliosides and modify the course of HAV infection in vivo. Miglustat, given by gavage to Ifnar1-/- mice (4,800 mg/kg of body weight/day) depleted hepatic gangliosides by 69 to 75% but caused substantial gastrointestinal toxicity and failed to prevent viral infection. UV-4, similarly administered in high doses (400 mg/kg/day), was well tolerated but depleted hepatic gangliosides by only 20% after 14 days. UV-4 depletion of gangliosides varied by class. Several GM2 species were paradoxically increased, likely due to inhibition of ß-glucosidases that degrade gangliosides. Both compounds enhanced, rather than reduced, virus replication. Nonetheless, both iminosugars had surprising anti-inflammatory effects, blocking the accumulation of inflammatory cells within the liver. UV-4 treatment also resulted in a decrease in serum alanine aminotransferase (ALT) elevations associated with acute hepatitis A. These anti-inflammatory effects may result from iminosugar inhibition of cellular α-glucosidases, leading to impaired maturation of glycan moieties of chemokine and cytokine receptors, and point to the potential importance of paracrine signaling in the pathogenesis of acute hepatitis A. IMPORTANCE Hepatitis A virus (HAV) is a common cause of viral hepatitis. Iminosugar compounds block its replication in cultured cells by inhibiting the synthesis of gangliosides required for HAV cell entry but have not been tested for their ability to prevent or treat hepatitis A in vivo. We show that high doses of the iminosugars miglustat and UV-4 fail to deplete gangliosides sufficiently to block HAV infection in mice lacking a key interferon receptor. These compounds nonetheless have striking anti-inflammatory effects on the HAV-infected liver, reducing the severity of hepatitis despite enhancing chemokine and cytokine expression resulting from hepatocyte-intrinsic antiviral responses. We propose that iminosugar inhibition of cellular α-glucosidases impairs the maturation of glycan moieties of chemokine and cytokine receptors required for effective signaling. These data highlight the potential importance of paracrine signaling pathways in the inflammatory response to HAV and add to our understanding of HAV pathogenesis in mice.

Modeling CNS Involvement in Pompe Disease Using Neural Stem Cells Generated from Patient-Derived Induced Pluripotent Stem Cells.

Pompe disease is a lysosomal storage disorder caused by autosomal recessive mutations in the acid alpha-glucosidase (GAA) gene. Acid alpha-glucosidase deficiency leads to abnormal glycogen accumulation in patient cells. Given the increasing evidence of central nervous system (CNS) involvement in classic infantile Pompe disease, we used neural stem cells, differentiated from patient induced pluripotent stem cells, to model the neuronal phenotype of Pompe disease. These Pompe neural stem cells exhibited disease-related phenotypes including glycogen accumulation, increased lysosomal staining, and secondary lipid buildup. These morphological phenotypes in patient neural stem cells provided a tool for drug efficacy evaluation. Two potential therapeutic agents, hydroxypropyl-ß-cyclodextrin and δ-tocopherol, were tested along with recombinant human acid alpha-glucosidase (rhGAA) in this cell-based Pompe model. Treatment with rhGAA reduced LysoTracker staining in Pompe neural stem cells, indicating reduced lysosome size. Additionally, treatment of diseased neural stem cells with the combination of hydroxypropyl-ß-cyclodextrin and δ-tocopherol significantly reduced the disease phenotypes. These results demonstrated patient-derived Pompe neural stem cells could be used as a model to study disease pathogenesis, to evaluate drug efficacy, and to screen compounds for drug discovery in the context of correcting CNS defects.

Effects of higher and more frequent dosing of alglucosidase alfa and immunomodulation on long-term clinical outcome of classic infantile Pompe patients.

The aim of this study was to compare the long-term outcome of classic infantile Pompe patients treated with 20 mg/kg alglucosidase alfa every other week (eow) to those treated with 40 mg/kg/week, and to study the additional effect of immunomodulation. Six patients received 20 mg/kg eow and twelve 40 mg/kg/week. Five patients were cross-reactive immunologic material (CRIM)-negative, two in the 20 mg, three in the 40 mg group. We compared (ventilator-free) survival, motor outcome, infusion associated reactions (IARs), and antibody formation. From 2012 on patients >2 months in the 40 mg group also received immunomodulation with rituximab, methotrexate, and intravenous immunoglobulin (IVIG) in an enzyme replacement therapy (ERT)-naïve setting. Survival was 66% in the 20 mg group and 92% in the 40 mg group. Ventilator-free survival was 50% and 92%. Both CRIM-negative patients in the 20 mg group died, whereas all three are alive in the 40 mg group. In the 20 mg group, 67% learned to walk compared with 92% in the 40 mg group. At the age of 3 years, 33% and 92% were able to walk. Peak antibody titers ranged from 1:1250 to 1:31 250 in the 20 mg group and from 1:250 to 1:800 000 in the 40 mg group. Five patients of the 40 mg group of whom two CRIM-negative also received immunomodulation. B-cell recovery was observed between 5.7 and 7.9 months after the last dose of rituximab. After B-cell recovery titers of patients with and without immunomodulation were similar (ranges 1:6 250-1:800 000 and 1:250-1:781 250). This study shows that classic infantile patients treated with 40 mg/kg/week from the start to end have a better (ventilator-free) survival and motor outcome. Immunomodulation did not prevent antibody formation in our study.

Moss-Derived Human Recombinant GAA Provides an Optimized Enzyme Uptake in Differentiated Human Muscle Cells of Pompe Disease.

Pompe disease is an autosomal recessive lysosomal storage disorder (LSD) caused by deficiency of lysosomal acid alpha-glucosidase (GAA). The result of the GAA deficiency is a ubiquitous lysosomal and non-lysosomal accumulation of glycogen. The most affected tissues are heart, skeletal muscle, liver, and the nervous system. Replacement therapy with the currently approved enzyme relies on M6P-mediated endocytosis. However, therapeutic outcomes still leave room for improvement, especially with regard to skeletal muscles. We tested the uptake, activity, and effect on glucose metabolism of a non-phosphorylated recombinant human GAA produced in moss (moss-GAA). Three variants of moss-GAA differing in glycosylation pattern have been analyzed: two with terminal mannose residues in a paucimannosidic (Man3) or high-mannose (Man 5) configuration and one with terminal N-acetylglucosamine residues (GnGn). Compared to alglucosidase alfa the moss-GAA GnGn variant showed increased uptake in differentiated myotubes. Moreover, incubation of immortalized muscle cells of Gaa-/- mice with moss-GAA GnGn led to similarly efficient clearance of accumulated glycogen as with alglucosidase alfa. These initial data suggest that M6P-residues might not always be necessary for the cellular uptake in enzyme replacement therapy (ERT) and indicate the potential of moss-GAA GnGn as novel alternative drug for targeting skeletal muscle in Pompe patients.

Potential Antidiabetic Activity of Extracts and Isolated Compound from Adenosma bracteosum (Bonati).

Adenosma bracteosum Bonati. (A. bracteosum) has been used in traditional and modern medicine in Vietnam for curing hepatitis. In this study, ethanol and aqueous extracts of A. bracteosum were evaluated for their α-glucosidase inhibitory activities and anti-hyperglycemic effects on glucose loaded hyperglycemic and streptozotocin (STZ) induced diabetic mice. The α-glucosidase inhibition of the extracts was evaluated by colorimetric assays, and the anti-diabetic activity was tested on a STZ-induced diabetic mice model. The ethanol and aqueous extracts showed a significant α-glucosidase inhibitory activity, which was more effective than acarbose at the same concentration. In the STZ-induced diabetic mice, both extracts showed a strong anti-hyperglycemic activity, with the group receiving 50 mg/kg of ethanol extract and the group receiving 50 mg/kg of aqueous extract presenting 64.42% and 57.69% reductions, respectively, in the blood glucose levels when compared with the diabetic control group, on day 21 (p > 0.05). Isoscutellarein-8-O-ß-D-glucopyranoside (IG) was identified from the ethanol extract, which showed a strong inhibitory activity against α-glucosidase, with a ten times higher potency compared with the positive control acarbose. The anti-hyperglycemic effect of IG was effectively similar to the standard drug, glibenclamide, at the same dose of 10 mg/kg (p > 0.05). These results indicated that A. bracteosum has a great antidiabetic potential.

Structural characterization and α-glycosidase inhibitory activity of a novel polysaccharide fraction from Aconitum coreanum.

α-Glycosidase is an essential target for the management of postprandial serum glucose in diabetic patients. Therefore, the interest has been growing in the screening of α-glycosidase inhibitor from natural resource. In the present study, the structure and α-glycosidase inhibitory activity of a polysaccharide (named as ACPP-1) from Aconitum coreanum were investigated. Based on the results from high performance gel permeation chromatography, GC-MS and 1D/2D nuclear magnetic resonance spectroscopy, ACPP-1 was a highly-linear polysaccharide with a molecular weight of 34.0 kD and containing over 90 % of glucose. It was composed of (1→4)-α-d-Glcp and α-Araf. ACPP-1 exhibited a dose-dependent inhibitory eff ;ect against α-glycosidase activity in vitro and the IC50 value was ∼0.8 mg/mL. In oral starch tolerance test, treatment with ACPP-1 (800 mg/kg) significantly improved the starch tolerance in mice. Taken together, this study provided a potential intervention and management for postprandial hyperglycemia by the polysaccharide fraction from A. coreanum.