Anti-enterovirus 71 activity of native fucosylated chondroitin sulfates and their derivatives.

Enterovirus 71 (EV71) is recognized as a major causative agent of hand, foot, and mouth disease (HFMD), posing a significant global public health concern due to its widespread impact and resulting in a major public health issue worldwide. Despite its prevalence, current clinical therapy lacks effective antiviral agents. Fucosylated chondroitin sulfates (FCS) derived from sea cucumber exhibits a range of biological activities including potent antiviral effects. This study provides compelling evidence of the potent antiviral efficacy of FCS against EV71. To further elucidate the impact of structural variations on the anti-EV71 activity, native FCSs with diverse sulfation patterns and a varity of FCS derivatives were prepared and analyzed. Notably, this study presents the detailed structural characterization of FCSs from the sea cucumbers Holothuria scabra Jaege and Holothuria fuscopunctata. Analysis of the structure-activity relationships revealed that molecular weight, sulfated fucose branches, and sulfation pattern were all crucial factors contributing to the potent inhibitory effects of FCS against EV71. Interestingly, molecular weight emerged as the most significant structural determinant of the antiviral potency. These findings suggest the promising potential of utilizing FCS as an innovative EV71 entry inhibitor for the treatment of HFMD.

Eco-friendly ozonation of alginate: Physicochemical characterization and degradation mechanism exploration through mass spectrometry.

The ozone degradation has been proven to be an effective degradation method for alginate, while the degradation mechanism remained to be unconfirmed. In this study, two high-molecular-weight alginates with different mannuronic/guluronic (G/M) ratios, HM and HG (G/M 0.49 vs 1.40), were depolymerized using established ozonation technology platform. Notably, HM can be degraded faster than HG especially within initial 30 min, indicating that the ß-1, 4-mannuronic bonds are more susceptible to be ozonated than α-1, 4-guluronic bonds. However, HM/HG degraded to LMWA in 2 h and reached a plateau. Therefore, we employed mass spectrometry (MS) to profile the degraded products of LMWA polymannuronate (PM) and polyguluronate (PG) in more intense conditions. The results indicated that the oxidation process continued until all reducing ends were converted to carboxyl groups. The o-diol could directly oxidize to o-dialdehyde. This study provides a MS based elucidation of the mechanism by which alginate cleaves to oligosaccharides through ozonation.

Structural Elucidation of Glycosaminoglycans in the Tissue of Flounder and Isolation of Chondroitin Sulfate C.

Glycosaminoglycans (GAGs) are valuable bioactive polysaccharides with promising biomedical and pharmaceutical applications. In this study, we analyzed GAGs using HPLC-MS/MS from the bone (B), muscle (M), skin (S), and viscera (V) of Scophthalmus maximus (SM), Paralichthysi (P), Limanda ferruginea (LF), Cleisthenes herzensteini (G), Platichthys bicoloratus (PB), Pleuronichthys cornutus (PC), and Cleisthenes herzensteini (CH). Unsaturated disaccharide products were obtained by enzymatic hydrolysis of the GAGs and subjected to compositional analysis of chondroitin sulfate (CS), heparin sulfate (HS), and hyaluronic acid (HA), including the sulfation degree of CS and HS, as well as the content of each GAG. The contents of GAGs in the tissues and the sulfation degree differed significantly among the fish. The bone of S. maximus contained more than 12 µg of CS per mg of dry tissue. Although the fish typically contained high levels of CSA (CS-4S), some fish bone tissue exhibited elevated levels of CSC (CS-6S). The HS content was found to range from 10-150 ug/g, primarily distributed in viscera, with a predominant non-sulfated structure (HS-0S). The structure of HA is well-defined without sulfation modification. These analytical results are independent of biological classification. We provide a high-throughput rapid detection method for tissue samples using HPLC-MS/MS to rapidly screen ideal sources of GAG. On this basis, four kinds of CS were prepared and purified from flounder bone, and their molecular weight was determined to be 23-28 kDa by HPGPC-MALLS, and the disaccharide component unit was dominated by CS-6S, which is a potential substitute for CSC derived from shark cartilage.

The role of corneal endothelium in macular corneal dystrophy development and recurrence.

Macular corneal dystrophy (MCD) is a progressive, bilateral stromal dystrophic disease that arises from mutations in carbohydrate sulfotransferase 6 (CHST6). Corneal transplantation is the ultimate therapeutic solution for MCD patients. Unfortunately, postoperative recurrence remains a significant challenge. We conducted a retrospective review of a clinical cohort comprising 102 MCD patients with 124 eyes that underwent either penetrating keratoplasty (PKP) or deep anterior lamellar keratoplasty (DALK). Our results revealed that the recurrence rate was nearly three times higher in the DALK group (39.13%, 9/23 eyes) compared with the PKP group (10.89%, 11/101 eyes), suggesting that surgical replacement of the corneal endothelium for treating MCD is advisable to prevent postoperative recurrence. Our experimental data confirmed the robust mRNA and protein expression of CHST6 in human corneal endothelium and the rodent homolog CHST5 in mouse endothelium. Selective knockdown of wild-type Chst5 in mouse corneal endothelium (ACsiChst5), but not in the corneal stroma, induced experimental MCD with similar extracellular matrix synthesis impairments and corneal thinning as observed in MCD patients. Mice carrying Chst5 point mutation also recapitulated clinical phenotypes of MCD, along with corneal endothelial abnormalities. Intracameral injection of wild-type Chst5 rescued the corneal impairments in ACsiChst5 mice and retarded the disease progression in Chst5 mutant mice. Overall, our study provides new mechanistic insights and therapeutic approaches for MCD treatment by high-lighting the role of corneal endothelium in MCD development.

Serum-based Comprehensive N-Glycans Profiling Analysis in Different Gastric Disease Stages by Porous Graphitic Carbon Liquid Chromatography-Mass Spectrometry Associated With Potential Marker Discovery.

BACKGROUND/AIM: N-glycans are potential serum biomarkers due to their aberrant structure and abundance alteration during disease progression. Few studies have been associated with relative quantitative N-glycans profiling during different gastric disease stages. In this study, we conducted an investigation on the profiling of N-glycans in patients with gastric disease, as well as in healthy controls. MATERIALS AND METHODS: In this study, the porous graphitization carbon chromatography-high resolution Fourier transform mass spectrometry (PGC-FTMS) method was applied to assess comprehensive N-glycans profiling in patients at different stages of gastric disease, including gastritis, atrophic gastritis, gastric ulcer, gastric polyps, and gastric cancer. RESULTS: A total of 45 N-glycans (relative abundance >0.1%) were detected, and 9 N-glycans were found to be potential biomarkers for gastric disease detection. Along with the progression of gastric disease, the abundance of sialylated N-glycans increased, while that of core-fucosylated N-glycans decreased. Multivariate statistical analysis demonstrated that N-glycans profiling between gastritis and healthy controls had significant differences. The characteristic N-glycans distinguished gastric cancer from healthy controls, which had strong clinical diagnostic value. CONCLUSION: The relative quantitative profile of N-glycans in different gastric disease stages was revealed and serum N-glycans are proposed for distinguishing gastric disease stages in clinical application.

Fucoidan from Fucus vesiculosus prevents the loss of dopaminergic neurons by alleviating mitochondrial dysfunction through targeting ATP5F1a.

Parkinson's disease is a neurodegenerative disease that is characterized by the loss of dopaminergic neurons. Fucoidan, which has emerged as a neuroprotective agent, is a marine-origin sulfated polysaccharide enriched in brown algae and sea cucumbers. However, variations in structural characteristics exist among fucoidans derived from different sources, resulting in a wide spectrum of biological effects. It is urgent to find the fucoidan with the strongest neuroprotective effect, and the mechanism needs to be further explored. We isolated and purified four different fucoidan species with different chemical structures and found that Type II fucoidan from Fucus vesiculosus (FvF) significantly improved mitochondrial dysfunction, prevented neuronal apoptosis, reduced dopaminergic neuron loss, and improved motor deficits in an 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. Further mechanistic investigation revealed that the ATP5F1a protein is a key target responsible for alleviating mitochondrial dysfunction of FvF to exert neuroprotective effects. This study highlights the favorable properties of FvF for neuroprotection, making FvF a promising candidate for the treatment of PD.

Oxygen Saturation Ranges for Healthy Newborns within 2 h at Altitudes between 847 and 4,360 m: A Prospective Cohort Study.

INTRODUCTION: The partial oxygen pressure in the air decreases with increasing altitude. This study was designed to compare the pulse oxygen saturation (SpO2) among well full-term neonates at different altitudes during their first 2 h after birth and to establish cutoff values of SpO2 identifying hypoxemia between 30 and 120 min after birth. METHODS: A multisite prospective cohort study was conducted at five participating hospitals from the Chinese High Altitude Neonatal Medicine Alliance. Healthy full-term infants were recruited and divided into four groups based on the altitude. Preductal SpO2 was recorded at 10 min, 10-30 min, and 30-120 min after birth. The 2.5th percentile of the SpO2 distribution range was considered as the cutoff for identifying hypoxemia at each altitude. RESULTS: A total of 727 infants were eligible for analysis. The SpO2 of neonates at different altitudes increased with the time after birth. A higher altitude was associated with lower SpO2, especially Shangri-La (3,509 m) and Yushu (4,360 m). The cutoff SpO2 for identifying hypoxemia during 30-120 min after birth were 94% in Xishuangbanna (847 m), 92% in Kunming (1,983 m), 89% in Shangri-La (3,509 m), and 83% in Yushu (4,360 m). CONCLUSION: An increase in altitude, especially Shangri-La (3,509 m) and Yushu (4,360 m), had a significant impact on SpO2 among healthy full-term neonates during their first 2 h of life. Establishing the cutoff value of SpO2 for identifying hypoxemia during the early postnatal period serves to optimize the oxygen therapy at different altitudes.

Chaetomorpha linum polysaccharides alleviate NAFLD in mice by enhancing the PPARα/CPT-1/MCAD signaling.

BACKGROUND: Green algae contain many polysaccharides. However, there is no information on whether Chaetomorpha linum polysaccharides (CLP) can modulate lipid and glucose metabolism. MATERIAL AND METHODS: CLP were extracted from chlorella and their components were characterized. Male C57BL/6 mice were randomized and provided with control chow as the control, or high fat diet (HFD) to induce nonalcoholic fatty liver disease (NAFLD). NAFLD mice were treated orally with water as the HFD group or with 50 or 150 mg/kg CLP daily for 10 weeks. The impact of CLP treatment on lipid and glucose metabolism and the PPARα signaling was examined by histology, Western blotting and biochemistry. RESULTS: CLP mainly contained arabinogalactan sulfate. Compared with the control, HFD feeding increased body weights, lipid droplet liver deposition and induced hyperlipidemia, liver functional impairment and glucose intolerance in mice. Treatment with CLP, particularly with a higher dose of CLP, limited the HFD-increased body weights and liver lipid droplet deposition, mitigated the HFD-induced hyperlipidemia and improved liver function and glucose tolerance in mice. Mechanistically, feeding with HFD dramatically decreased the expression of liver PPARα, CPT-1, and MCAD, but treatment with CLP enhanced their expression in a trend of dose-dependent in mice. CONCLUSIONS: These findings indicated that CLP treatment alleviated the gain in body weights, NAFLD, and glucose intolerance in mice after HFD feeding by enhancing the PPARα/CPT-1/MCAD signaling.

The dose-response relationship between sex hormones and hyperuricemia in different gender: NHANES 2013-2016.

Objectives: To access the dose-response relationship between sex hormones and hyperuricemia (HUA), and to find the cut-off value in different gender. Methods: 9,685 participants were derived from the database of National Health and Nutrition Examination Survey (NHANES). Restricted cubic spline (RCS) analysis were applied to explore the relationship between sex hormones and HUA after adjusting for confounding factors by propensity score match (PSM). Logistic regression was used to estimate the odds ratio (OR) and 95% CI. Results: The prevalence of HUA was 15.13% in female participants and 22.30% in male participants. Logistic regression analysis showed that estradiol (E2) was independently associated with HUA for a P value of 0.003 and 0.01in female and male participants, respectively. Testosterone (T) was only independently associated with HUA in male participants (P<0.001) but not in female participants (P = 0.59). RCS analysis showed a dose-response relationship between sex hormones and HUA. The risk of HUA increased as E2 lower than 29.6pg/mL in female participants and T lower than 389.1ng/dL in male participants. E2 higher than 23.6pg/ml was an independent risk factor for HUA in male participants. Conclusion: A dose-response relationship was found between sex hormones and HUA. The cut-off value of E2 in male and female participants was 29.6pg/mL and 23.6pg/mL, respectively, and the cut-off value of T in male participants was 389.1ng/dL. These results provide a reference for preventing HUA and hormone supplement therapy.

Pain assessment during physiotherapy and noxious stimuli in patients with disorders of consciousness: A preliminary study.

Objectives: The primary purpose of this study is to determine whether patients with disorders of consciousness (DOC) (unresponsive wakefulness syndrome, UWS; minimally conscious state, MCS) experience pain during physiotherapy and noxious stimuli in a larger patient population. Materials and methods: The patients' level of consciousness was measured with the Coma Recovery Scale-Revised (CRS-R). Additionally, the Nociception Coma Scale-revised (NCS-R) was used to assess their pain response. The NCS-R total scores between UWS and MCS at baseline, physiotherapy and noxious stimulus were compared using the Mann-Whitney U test (Wilcoxon rank-sum test) and the Kruskal-Wallis H test with Bonferroni correction. Results: The study enrolled 93 participants. There was a statistically significant difference in NCS-R total scores between the three conditions (H = 215.25, p < 0.001). At baseline, there was no statistically significant difference between MCS and UWS (U = 378, z = -1.35, p = 0.178). While there was a statistically significant difference between MCS and UWS during physiotherapy (U = 1,362, z = -3.06, p < 0.01) and under noxious stimuli (U = 5142.5, z = -11.22, p < 0.001). Conclusion: Physiotherapy improved the activity responsiveness of DOC patients, and patients experienced less potential pain. However, some DOC patients, especially MCS patients, perceived pain under the noxious stimuli.

Canagliflozin Prevents Lipid Accumulation, Mitochondrial Dysfunction, and Gut Microbiota Dysbiosis in Mice With Diabetic Cardiovascular Disease.

Type 2 diabetes mellitus (T2DM) is associated with cardiovascular disease (CVD) and sodium glucose cotransporter 2 inhibitors, as oral medications for T2DM treatment have shown the potential to improve vascular dysfunction. The aim of this study was to evaluate the ability of canagliflozin (Cana) to relieve CVD in T2DM mice and its possible action mechanism. Mice with diabetic CVD was conducted by a high-fat diet for 24 weeks, followed by oral gavaging with metformin (200 mg/kg/day) or Cana (50 mg/kg/day) for 6 weeks. The result demonstrated that Cana reduced serum lipid accumulation, and decreased the arteriosclerosis index and atherogenic index of plasma. In addition, Cana treatment reduced the circulating markers of inflammation. More importantly, Cana improved cardiac mitochondrial homeostasis and relieved oxidative stress. Moreover, Cana treatment alleviated the myocardial injury with decreasing levels of serous soluble cluster of differentiation 40 ligand and cardiac troponin I. Thus, cardiovascular abnormality was relieved by suppressing fibrosis and basement membrane thickening, while elevating the cluster of differentiation 31 expression level. Importantly, Cana increased the ratio of gut bacteria Firmicutes/Bacteroidetes and the relative abundance of Alistipes, Olsenella, and Alloprevotella, while it decreased the abundance of Mucispirillum, Helicobacter, and Proteobacteria at various taxonomic levels in mice with diabetic CVD. In short, Cana treatment altered the colonic microbiota composition close to the normal level, which was related with blood lipid, inflammation, and oxidative stress, and might play a vital role in CVD. In general, the improvements in the gut microbiota and myocardial mitochondrial homeostasis may represent the mechanism of Cana on CVD treatment.

Biochemical Properties of a New Polysaccharide Lyase Family 25 Ulvan Lyase TsUly25B from Marine Bacterium Thalassomonas sp. LD5.

Marine macroalgae, contributing much to the bioeconomy, have inspired tremendous attention as sustainable raw materials. Ulvan, as one of the main structural components of green algae cell walls, can be degraded by ulvan lyase through the ß-elimination mechanism to obtain oligosaccharides exhibiting several good physiological activities. Only a few ulvan lyases have been characterized until now. This thesis explores the properties of a new polysaccharide lyase family 25 ulvan lyase TsUly25B from the marine bacterium Thalassomonas sp. LD5. Its protein molecular weight was 54.54 KDa, and it was most active under the conditions of 60 °C and pH 9.0. The Km and kcat values were 1.01 ± 0.05 mg/mL and 10.52 ± 0.28 s-1, respectively. TsUly25B was salt-tolerant and NaCl can significantly improve its thermal stability. Over 80% of activity can be preserved after being incubated at 30 °C for two days when the concentration of NaCl in the solution is above 1 M, while 60% can be preserved after incubation at 40 °C for 10 h with 2 M NaCl. TsUly25B adopted an endolytic manner to degrade ulvan polysaccharides, and the main end-products were unsaturated ulvan disaccharides and tetrasaccharides. In conclusion, our research enriches the ulvan lyase library and advances the utilization of ulvan lyases in further fundamental research as well as ulvan oligosaccharides production.

Targeting lectin-like oxidized low-density lipoprotein receptor-1 triggers autophagic program in esophageal cancer.

Autophagy is a highly conserved catabolic process to maintain cellular homeostasis. However, dysfunctional autophagy contributes to a context-dependent role in cancer. Here, we clarified the exact role of autophagy modulated by the scavenger receptor lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in esophageal cancer (EC). A comprehensive analysis in various cancers displayed that LOX-1 was upregulated the most in EC tissues and associated with poor prognosis of patients. Deletion of LOX-1 ex vivo and in vivo suppresses EC development by inducing autophagic cell death. Receptor for activated C kinase 1 (RACK1) was identified as a signal adapter of LOX-1, which incented RAS/MEK/ERK pathway and TFEB nuclear export signal and safeguarded tumorigenesis. A sulfated polysaccharide fucoidan extracted from brown seaweed was found to bind with LOX-1 and mediate its proteasomal degradation but not the lysosome pathway, leading to autophagy-related cell death in EC. These results reveal a central contribution of LOX-1 to EC development and provide genetic ablation or bioactive polysaccharide as an effective intervention for EC therapy.

Comparison of Different Labeling Techniques for the LC-MS Profiling of Human Milk Oligosaccharides.

Human milk oligosaccharides (HMOs) exhibit various biological activities for infants, such as serving as prebiotics, blocking pathogens, and aiding in brain development. HMOs are a complex mixture of hetero-oligosaccharides that are generally highly branched, containing multiple structural isomers and no intrinsic chromophores, presenting a challenge to both their resolution and quantitative detection. While liquid chromatography-mass spectrometry (LC-MS) has become the primary strategy for analysis of various compounds, the very polar and chromophore-free properties of native glycans hinder their separation in LC and ionization in MS. Various labeling approaches have been developed to achieve separation of glycans with higher resolution and greater sensitivity of detection. Here, we compared five commonly used labeling techniques [by 2-aminobenzamide, 2-aminopyridine, 2-aminobenzoic acid (2-AA), 2,6-diaminopyridine, and 1-phenyl-3-methyl-5-pyrazolone] for analyzing HMOs specifically under hydrophilic-interaction chromatography-mass spectrometry (HILIC-MS) conditions. The 2-AA labeling showed the most consistent deprotonated molecular ions, the enhanced sensitivity with the least structural selectivity, and the sequencing-informative tandem MS fragmentation spectra for the widest range of HMOs; therefore, this labeling technique was selected for further optimization under the porous graphitized carbon chromatography-mass spectrometry (PGC-MS) conditions. The combination strategy of 2-AA labeling and PGC-MS techniques provided online decontamination (removal of excess 2-AA, salts, and lactose) and resolute detection of many HMOs, enabling us to characterize the profiles of complicated HMO mixtures comprehensively in a simple protocol.

Highly sialylated mucin-type glycopeptide from porcine intestinal mucosa after heparin extraction: O-glycan profiling and immunological activity evaluation.

Mucins are the major proteins that distributed on the intestinal mucosa layer and protect the intestine from pathogens infection. The composition of intestinal mucin O-glycans can affect the health of the gastrointestinal tract in pigs. Porcine intestinal mucosa is widely used as the main raw material of heparin extraction. The heparin extraction residues rich in mucins were usually wasted. The structure of mucin derived O-glycans in porcine intestinal mucosa are currently unknown. In this study, we isolated the mucins from the heparin extraction residues and profiled the O-glycans. After heparin extraction, mucin was digested with trypsin, and separated by strong anion exchange chromatography. The mucin derived O-glycans were release by alkaline ß elimination, and analyzed by ultra high performance liquid chromatography-porous graphitized carbon-Fourier transform mass spectrometry (UPLC-PGC-FTMS/MS). Thirty five kinds of O-glycans were identified, most of which were Core 3-derived glycans. In particular, the O-glycans containing sialic acid Neu5Ac accounted for 71.93% of the total O-glycans, which were different from that of other species, including mouse intestine, fish intestine, and porcine colon. The high content sialylated mucin may explain its effect in biological processes. Furthermore, the immunological activity results indicated that the porcine intestinal mucin could promote phagocytosis and proliferation without any cytotoxic effects, which may aid in the development of immunomodulators.

In vitro fermentation of hyaluronan by human gut microbiota: Changes in microbiota community and potential degradation mechanism.

Hyaluronan (HA) has been widely used as a dietary supplement which can be degraded by gut microbiota. However, the interactions between HA and gut microbiota have not been fully characterized. Here, using an in vitro system, we found that HA is readily fermented by human gut microbiota but with differing fermentative activities among individuals. HA-fermentation boosted Bacteroides spp., Bifidobacterium spp., Dialister spp., Faecalibacterium spp. and produced a significant amount of acetate, propionate and butyrate. Fermentation products profiling indicated that HA could be degraded into unsaturated even-numbered and saturated odd-numbered oligosaccharides. Further, polysaccharide lyases (PLs) and glycoside hydrolases (GHs) including GH88, PL8, PL29, PL35 and PL33 were identified from B. ovatus E3, which can help to explain the structure of the fermentation products. Collectively, our study sheds new light into the metabolism of HA and forms the basis for understanding the bioavailability of HA from a gut microbiota perspective.

Therapeutic Effect of Idebenone on Rats with Vascular Dementia via the MicroRNA-216a/RSK2/NF-κB Axis.

BACKGROUND: Vascular dementia (VD) is a brain disease featured by cognitive impairment and cerebrovascular pathologies. Idebenone can treat neurodegenerative diseases. This study evaluated the mechanism of Idebenone in VD. METHODS: The VD rat model was established by permanent occlusion of bilateral common carotid arteries, followed by intragastrical administration of Idebenone. The learning and spatial memory abilities, and the levels of MDA, SOD, IL-6 and TNF-α were measured. Histological staining was adopted to observe the damage of neurons in the hippocampal cortex and to quantitatively analyze the neuronal damage in CA1 area of hippocampus. Microarray analysis was performed to find out the effect of Idebenone treatment on microRNA (miR) expression in hippocampus of rats. The potential target genes of miR and the pathways regulated by target genes were searched by bioinformatics analysis, and verified by experiments. The mechanism of action behind Idebenone in VD rats was proved by rescue experiment. RESULTS: Idebenone treatment improved the learning and spatial memory abilities of VD rats, inhibited neuroinflammation and oxidative stress, and prevented neuronal apoptosis. Idebenone treatment elevated miR-216a expression in hippocampus of rats, but the therapeutic effect of Idebenone was averted by lentivirus inhibition of miR-216a. miR-216a targeted RSK2. Overexpression of RSK2 annulled the therapeutic effect of Idebenone on VD rats by activating the IκBα/NF-κB axis. CONCLUSION: Idebenone inhibits the activation of RSK2/IκBα/NF-κB axis by increasing miR-216a, thus alleviating oxidative stress and neuroinflammation in VD rats.

Recent progress and advanced technology in carbohydrate-based drug development.

Carbohydrates, one of the most abundant and widespread biomolecules in nature, play indispensable roles in diverse biological functions, and represent a treasure trove of untapped potential for pharmaceutical applications. Here, we provide a brief overview of carbohydrate-based drug development (CBDD) over the past two decades. More importantly, advanced techniques and methodologies related to CBDD are emerging, including enzymatic synthesis, metabolic engineering, site-specific glycoconjugation, carbohydrate libraries and microarrays as well as carbohydrate-gut microbiome evaluation. These technologies have dramatically accelerated the speed of CBDD. The recently approved drugs and emerging techniques summarized herein will inspire new sights into potential opportunities to discover novel carbohydrate drugs.